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Specific nutrient removal rates in saline wastewater treatment using sequencing batch reactor
Process Biochemistry 41 (2006) 61–66 www.elsevier.com/locate/procbio
Specific nutrient removal rates in saline wastewater treatment using sequencing batch reactor Ahmet Uygur * Department of Environmental Engineering, Uludag˘ Universiy, Go¨ru¨kle, Bursa, Turkey Received 21 October 2004; received in revised form 26 January 2005; accepted 12 March 2005
Abstract Effects of salt concentration (0–6%, w/v) on specific nutrient removal rates from saline wastewater in a sequencing batch reactor (SBR) were investigated. The sequencing batch operation consisted of anaerobic, oxic, anoxic and oxic phases with hydraulic residence times (HRT) of 1/3/1/1 h and a settling phase of 3/4 h. Solids retention time (SRT) was kept constant at 10 days in all experiments. Specific nutrient (COD, NH4-N and PO4-P) removal rates decreased with increasing salt concentration due to adverse effects of salt on microorganisms. A salt tolerant organism, Halobacter halobium was added to the activated sludge culture (1/1, v/v) in order to improve the nutrient removal performance of the SBR. Nutrient removal performances of Halobacter-free and Halobacter-added activated sludge cultures were compared for all salt contents tested. Specific rates of nutrient removal obtained with the Halobacter-added culture were higher that those of Halobacter-free activated sludge, especially at high salt contents. # 2005 Elsevier Ltd. All rights reserved. Keywords: Halobacter halobium; Saline wastewater; Nutrient removal; Sequencing batch reactor (SBR)
1. Introduction Biological treatment of saline wastewater usually results in low BOD/COD removal performances because of adverse effects of salt on microbial flora. High salt concentration (>1% salt) causes plasmolysis and loss of activity of the cells. Several researchers have investigated the effects of salinity on bioactivity of microorganisms in biological treatment systems and found that high levels of salinity had adverse effects on treatment performance [1–3]. Matsuo and Hosobora [4] studied the effect of salinity on enhanced biological phosphorus removal and reported that NaCl concentrations above 20 mM (1.17 g l1) restricted the luxury phosphorus uptake resulting in high phosphorus contents in the effluent. However, the system was able to resume its performance after an appropriate acclimation period. Panswad and Anan [5,6] observed an improvement in the carbon and nitrogen removal efficiency in biological nutrient removal from high salinity wastewater when seeded * Tel.: +90 224 4428177; fax: +90 224 4429148. E-mail address: [email protected]. 1359-5113/$ – see front matter # 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.procbio.2005.03.068
with salt-acclimated cultures. Phosphorus-accumulating bacteria were found to be more sensitive to the high salt concentrations than nitrifiers and denitrifiers. Dahl et al. [7] investigated nitrification and denitrification of gas scrubbing liquors which have high chloride content. Maximum rates of nitrification and denitrification were reported as 2 and 3 mgN gVSS1 h1, respectively, at 30 8C and 20 g Cl l1 chloride concentration. Yang et al. [8] investigated biological treatment of brine wastewater generated by ion exchange processes. Because of utilization of entrapped mixed culture, no significant salt inhibition of nitrate removal was observed up to 10 g l1 (1%) salt concentrations. Salt inhibition effects on nitrification of synthetic wastewater were investigated by Kargi and Dincer using an activated sludge unit [9]. Experiments performed with 3% salt content at different sludge ages were used for determination of salt inhibition constants for nitrification. A non-competitive inhibition affecting both the maximum rate and the saturation constants were found to be suitable for salt inhibition of nitrification. Numerous studies in the literature have reported on biological nutrient removal [10–18]. Kargi and Uygur used a
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five-step SBR for nutrient removal from a synthetic wastewater. Effects of the sludge age (SRT), hydraulic residence time (HRT), feed wastewater composition and carbon sources on nutrient removal performance were investigated. Sludge age of 10 days and a wastewater composition of COD/N/P = 100/3.33/0.69 were determined as optimal values resulting in maximum percent nutrient removal [19,20]. COD removal from saline wastewater was investigated indicating salt inhibition at salt concentrations above 2% [21–24]. However, only a few studies are reported in literature on biological nutrient (COD, N, P) removal from low salinity (salt < 1%) wastewater. Intrasungkha et al. [25] investigated biological nutrient removal from saline wastewater using an SBR with satisfactory nutrient removals at low salt contents (0.03–0.2% NaCl). Abu-ghararah and Sherrard [26] determined that salinity had little effect on nitrogen removal at 0.4% salt content (4 g/l NaCl). However, percent phosphorus removal decreased from 82% with saltfree wastewater to 25 with 0.4% salt. Dan et al. [27] used an osmo-tolerant yeast culture for biological treatment of saline wastewater (2–4.5% NaCl) and compared the treatment performance with that of the bacterial culture. Specific nutrient uptake for the yeast cells was found to be higher than that of the bacterial culture resulting in higher nitrogen and phosphorous removal rates. Effects of high salt contents on the extent of nutrient (COD, N, P) removal in an SBR was investigated for a large range of salt contents above 1% [28]. None of the aforementioned studies on saline wastewater treatment specific nutrient removal rates were investigated for a large range of salt concentrations using an SBR. Therefore, the major objective of this study is to investigate and alleviate the adverse effects of high salt contents on specific nutrient removal rate (SNRR) using an SBR. A fourstep SBR consisting of anaerobic/oxic/anoxic/oxic phases with hydraulic residence times (HRT) of 1/3/1/1 h and a settling phase of 3/4 h was used for COD, ammonium-N, phosphate-P removals from saline wastewater at different salt contents (0–6% salt). The sludge age (SRT) was kept constant at 10 days. In the first phase of the study the adverse effects of salt content on specific nutrient removal rates (SNRR) were investigated by using an activated sludge culture. In the second phase the same experiments were repeated using the salt tolerant bacteria, Halobacter-added (1/1, v/v) activated sludge culture to alleviate the adverse effect of salt on nutrient removal rates. Specific nutrient removal rates for both cultures were compared.
2. Materials and methods 2.1. Experimental set-up A schematic diagram of the experimental set up is shown in Fig. 1. A fermenter (Bioflo IIC, New Brunswick) with 5 l working volume was used as the sequencing batch reactor
Fig. 1. Schematic diagram of the sequencing batch reactor used in experimental studies.
(SBR). The fermenter was microprocessor controlled for aeration, agitation, pH and dissolved oxygen (DO). Aeration was provided by using an air pump and a sparger. Agitation speed was varied between 25 and 300 rpm. DO, pH, and ORP of the nutrient medium were continuously monitored by the relevant probes. 2.2. Wastewater composition Synthetic wastewater used throughout the studies was composed of glucose, sodium acetate, NH4Cl, KH2PO4, MgSO47H2O, NaHCO3 and certain trace minerals such as KCl (20 mg l1), CaCl22H2O (50 mg l1) and FeCl36H20 (50 mg l1). Salt (NaCl) concentration was varied between 0 and 6% (w/v). Typical composition of the synthetic wastewater was COD0 = 1200 50 mg l1, NT = 60 3 mg l1 and PT=18 2 mg l1, resulting in COD/N/P = 100/5/1.5. MgSO4 and NaHCO3 concentrations in the feed were 0.1 and 0.708 g l1, respectively, throughout the studies. The pH of the synthetic wastewater was adjusted to 7.0 at the beginning. 2.3. Organisms A mixed microbial population composed of heterotrophic organisms capable of oxidizing carbonaceous compounds and denitrification; autotrophic nitrifying organisms; anaerobic acid producers and excess phosphate uptaking organisms (Acinetobacter sp.) were used as inoculum culture. Nitrification organisms (Nitrosomonas and Nitrobacter) were obtained from Clemson University, SC, USA. The heterotrophic mixed culture was obtained from Cigli Municipal Wastewater Treatment Plant, Izmir. Anaerobic acid producers were obtained from the first stage (acid formation reactor) of the anaerobic wastewater treatment plant of PAKMAYA Bakers Yeast Company, Izmir, Turkey. The culture was cultivated in laboratory and added to the
A. Uygur / Process Biochemistry 41 (2006) 61–66
inoculum culture. Acinetobacter calcoaceticus (NRRL-552) obtained from the USDA, National Research Laboratories, Peoria, IL, USA was used for excess phosphate uptake. Heterotrophic organisms were cultivated in the same synthetic nutrient media used in the experiments. The growth media for denitrifying organisms contained 800 mg l1 glucose, 50 mg l1 KH2PO4, 610 mg l1 NaNO3, 20 mg l1 MgSO47H2O and 10 mg l1 Na2S2O3 at pH 7. The growth media used for cultivation of nitrification organisms contained 382 mg l1 NH4Cl, 1510 mg l1 NaHCO3, 93 mg l1 glucose, 60 mg l1 KH2PO4, 20 mg l1 CaCl22H2O and 40 mg l1 MgSO47H2O at pH 7.5. The growth media for Acinetobacter calcoaceticus was composed of 5000 mg l1 glucose, 3000 mg l1 KH2PO4, 1000 mg l1 NH4Cl, 500 mg l1 yeast extract, 3000 mg l1 NaCl and 50 mg l1 MgSO47H2O at pH 7. The growth media for Halobacter halobium was composed of 8 g l1 glucose, 300 mg l1 KH2PO4, 1 g l1 NH4Cl, 250 mg l1 yeast extract, 30 g l1 NaCl (3%, w/v) and 50 mg l1 MgSO47H2O at pH 7.5. These cultures were cultivated in suitable growth media in the laboratory and were used as inocula in the form of a mixed culture. Halobacter halobium (ATCC 43214) was obtained from the American Type of Culture Collection (ATCC), Maryland, USA in lyophilized form and cultivated in salt containing synthetic media in the laboratory. Halobacter culture was mixed with activated sludge culture (1/1, v/v) for the experiments performed with the Halobacter-added activated sludge culture to overcome salt inhibition effects on the specific nutrient uptake rates. The concentration of the mixed microbial culture was approximately 3.14 g l1 at the beginning. 2.4. Experimental procedure The reactor was filled with the synthetic wastewater, inoculated with a mixed culture of microorganisms and was operated batchwise with aeration and mixing for several days to obtain a dense culture to start with. After sedimentation of the organisms, 4 l clear supernatant was removed and 1 l dense culture was completed to 5 l total volume with the defined nutrient medium. Then, anaerobic, oxic, anoxic and oxic operations were applied in sequence. Nitrogen gas was passed through the medium only during anaerobic operation. Agitation speed during anaerobic and anoxic cycles was 25 and 50 rpm, respectively. Anoxic conditions were provided by keeping the DO nearly zero and the ORP between 0 and 100 mV. The medium was aerated and agitated (300 rpm) vigorously during oxic (aerobic) operation. Oxidation reduction potentials (ORP) for the anaerobic, oxic, anoxic and oxic phases were approximately 250, +250, 70 and +250 mV, respectively. Samples were withdrawn from the reactor at the beginning and at the end of each cycle for analysis. At the end of each SBR operation, the organisms were settled for 3/4 h and 4 l of the supernatant wastewater was removed. Settled organisms
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of 1 l volume were used for the next treatment operation with the addition of 4 l fresh nutrient media. A fraction of (1/10) of the culture was removed from the reactor daily before settling to adjust the sludge age to 10 days. Temperature and pH was controlled around T = 25 1 8C and pH 7–7.5. Dissolved oxygen (DO) concentration in oxic (aerobic) phase was kept above 2 mg l1, while the DO levels during anaerobic and anoxic phases were practically zero. A four-step SBR operation consisting of anaerobic/oxic/ anoxic/oxic steps with HRT’s of 1/3/1/1 h was used at all salt concentrations. Every experiment was carried out four times at any salt content tested. Data were collected at the end of each phase for every experiment. The system reached the steady-state after 3 days of operation resulting in almost the same nutrient removal. Since the difference in results was negligible for the last two runs (<3% of the average), no further replicates were conducted. 2.5. Analytical methods Samples were withdrawn from the liquid media at the beginning and at the end of each treatment phase (anaerobic, oxic, anoxic, oxic) and were centrifuged at 6000 rpm for 30 min to remove microorganisms from the liquid medium. Clear supernatants were analyzed for COD, ammonium-N and phosphate-P contents. Standard kit (Merck-Spectroquant) and spectrometric method were used for ammoniumN and phosphate-P analyses. COD, total solids (TS) and total suspended solids (TSS) concentrations were determined by using the Standard Methods [29]. Samples were analyzed in triplicates and average values were reported. DO, pH and ORP measurements were done by using the relevant probes and analyzers. Samples were centrifuged to separate saline water from the biomass and the washed salt-free organisms were used in determining the biomass concentrations. Biomass concentrations were determined by filtering the washed salt-free samples through 0.45 mm membrane filter and drying in an oven at 105 8C to constant weight.
3. Results and discussion 3.1. Specific rate of COD removal Variation of specific removal rates of COD with the salt content is depicted in Fig. 2 for the Halobacter-free and Halobacter-added activated sludge cultures. The specific COD removal rate increased from 34.0 to 42.5 mg COD g biomass1 h1 when the salt content was increased from 0 to 1% for the Halobacter-free activated sludge culture indicating stimulatory effects of low salt contents (<1% salt) on the organisms. Further increases in salt content caused severe decreases in specific COD removal rate resulting in 26.7 and 10.7 mg COD g biomass1 h1. COD removal rates at 3 and 6% salt contents, respectively. Apparently, salt contents above 1% caused cell disintegra-
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Fig. 2. Variation of specific COD removal rate with salt content. (*) Halobacter-free activated sludge culture and (*) halobacter-added activated sludge culture.
tion resulting in lower specific COD removal rates in the activated sludge culture. Specific COD removal rates obtained with the Halobacter-added activated sludge was higher than those of Halobacter-free activated sludge especially at high salt contents. Specific COD removal rate increased from 42.6 to 63.3 mg COD g biomass1 h1 when salt content increased from 0 to 1% in the Halobacter-added activated sludge culture indicating stimulatory effects of low salt contents on the organisms. Further increases in salt content caused decreases in COD removal rates, however to a lesser extent as compared to the Halobacter-free activated sludge. The specific COD removal rate decreased to 44 mg COD g biomass1 h1 at 3% salt and further to 31.0 mg COD g biomass1 h1 at 6% salt content. Apparently, the salt tolerant bacteria, Halobacter halobium was not affected from high salt contents above 1% yielding high COD removal rates as compared to the activated sludge culture. Inclusion of Halobacter culture to the activated sludge was found to be beneficial by providing high COD removal rates. Data presented in Fig. 2 for the specific COD removal rate by the Halobacter-free and Halobacter-added activated sludge culture were correlated with a fourth-order polynomial equation in the following form. Halobacter-free activated sludge: RCOD ¼ 0:55 S4 þ 6:5 S3 24:1 S2 þ 23:75 S þ 36:6 2
ðR ¼ 0:90Þ
3.2. Specific rate of ammonium-N removal Variation of specific removal rate of NH4-N with salt content is depicted in Fig. 3 for the Halobacter-free and Halobacter-added activated sludge cultures. Specific NH4-N removal rate decreased with increasing salt content for both cultures. However, the Halobacter-added culture resulted in higher specific NH4-N removal rates as compared to the Halobacter-free activated sludge at all salt concentrations tested. The specific rate of NH4-N removal decreased from 3.0 to 0.80 mg NH4-N g biomass1 h1 when salt content increased from 0 to 6% for the Halobacter-free activated sludge culture. Halobacter-added activated sludge culture resulted in NH4-N removal rates of 3.04 and 1.45 mg NH4N g biomass1 h1 at 0 and 6% salt contents, respectively. The results indicated that Halobacter addition to the activated sludge culture improved the specific NH4-N removal rates significantly, especially at high salt concentrations. The data presented in Fig. 3 for the specific NH4-N removal rate were correlated with exponential equations in the following forms for both cultures. Halobacter-free activated sludge: RNH4 -N ¼ 3:024 e0:1978S
(1)
RCOD ¼ 0:46 S4 þ 6:3 S3 28:5 S2 þ 42:5 S þ 41:4
ðR2 ¼ 0:93Þ
(3)
Halobacter-added activated sludge: RNH4 -N ¼ 3:152 e0:1204S
Halobacter-added activated sludge:
ðR2 ¼ 0:98Þ
Fig. 3. Variation of specific NH4-N removal rate with salt content. (*) Halobacter-free activated sludge culture and (*) halobacter-added activated sludge culture.
ðR2 ¼ 0:93Þ
(4)
where, RNH4 -N is the specific rate of NH4-N removal (mg NH4-N g biomass1 h1) and (S) is the percent salt concentration. 3.3. Specific rate of PO4-P removal
(2)
where, RCOD is the specific COD removal rate mg COD g biomass1 h1 and (S) is the percent salt concentration.
Variation of specific PO4-P removal rates with the salt concentration are depicted in Fig. 4 for both Halobacter-free and Halobacter-added activated sludge culture. Specific rates of PO4-P removal decreased with increasing salt
A. Uygur / Process Biochemistry 41 (2006) 61–66
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4. Conclusions
Fig. 4. Variation of specific PO4-P removal rate with salt content. (*) Halobacter-free activated sludge culture and (*) halobacter-added activated sludge culture.
content for both cultures. However, the adverse effects of salt were more pronounced with the Halobacter-free activated sludge. The specific rate of PO4-P removal decreased 0.36–0.08 mg PO4-P g biomass1 h1 when salt content increased from 0 to 6% for the Halobacter-free activated sludge culture indicating severe adverse effects of high salt contents on phosphate removal rates. Halobacteradded activated sludge culture resulted in PO4-P removal rates of 0.52 and 0.18 mg PO4-P g biomass1 h1 at 0 and 6% salt contents, respectively. Specific rates of PO4-P removal obtained with Halobacter-added activated sludge culture were higher than those of Halobacter-free activated sludge at all salt contents tested. Plasmolysis of excess phosphate uptaking organisms and release of deposited intracellular phosphate compounds might be responsible for the low the specific PO4-P uptake rates at high salt contents with the Halobacter-free activated sludge. The results indicated clear advantage of using Halobacter in the activated sludge culture in order to obtain high PO4-P removal rates. Halobacter halobium species are known to be incapable of excess phosphate uptake. Therefore, the improvement in specific PO4-P uptake rate at high salt contents is probably because of phosphate assimilation by the Halobacter species for biosynthesis. The specific PO4-P removal rate data presented in Fig. 4 were correlated with exponential equations in the following form for both cultures. Halobacter-free activated sludge: RPO4 -P ¼ 0:282 e0:2278S
ðR2 ¼ 0:79Þ
(5)
Halobacter-added activated sludge: RPO4 -P ¼ 0:448 e0:1780S
ðR2 ¼ 0:89Þ
(6)
where, RPO4 -P is the specific PO4-P removal rate (mg PO4P g biomass1 h1) and (S) is the percent salt concentration.
Specific nutrient removal rates from saline wastewater were investigated using Halobacter-free and Halobacteradded activated sludge cultures in a sequencing batch reactor (SBR). A four-step SBR operation consisting of anaerobic/ oxic/anoxic/oxic steps with 1/3/1/1 h hydraulic residence times (HRT) and a constant sludge age of 10 days was used throughout the studies. Salt content of the synthetic wastewater was varied between 0 and 6%. Specific rates of nutrient removal decreased with increasing salt content for both cultures. However, nutrient removal rates were higher with the Halobacter-added activated sludge culture as compared to the Halobacter-free culture. Low contents of salt (<1%) were found to be stimulatory for the organisms resulting in an improved COD removal rate. Because of the plasmolysis of the activated sludge organisms at high salt contents, specific nutrient removal rates decreased with increasing salt content. Improved specific nutrient removal rates were obtained with the Halobacter-added activated sludge culture especially at salt contents above 1% (w/v), due to the salt tolerant characteristics of Halobacter halobium species. Specific COD, NH4-N and PO4-P removal rates were 10.7 mg COD g biomass1 h1, 0.80 mg NH4-N g biomass1 h1 and 0.08 mg PO4-P g biomass1 h1 for the Halobacterfree activated sludge culture while those values were 31.0 mg COD g biomass1 h1, 1.45 mg NH4-N g 1 1 biomass h and 0.18 mg PO4-P g biomass1 h1 for the Halobacter-added activated sludge. The results indicated clear advantage of inclusion of Halobacter halobium to the activated sludge in order to obtain high nutrient removal rates especially at high salt concentrations.
Acknowledgements The author expresses his gratitude to Prof. Dr. Fikret Kargi for his valuable contribution to the critical review of the paper. This study was supported by the research funds of Uludag University in Bursa and Dokuz Eylul University in Izmir, Turkey.
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[17] Chang HN, Ra KM, Byung GP, Seong-Jin L, Dong WC, Woo GL, et al. Simulation of sequential batch reactor (SBR) operation for simultaneous removal of nitrogen and phosphorus. Bioproc Eng 2000;23: 513–21. [18] Zuniga MAG. Martinez SG. Biological phosphate and nitrogen removal in a biofilm sequencing batch reactor. Water Sci Technol 1996;34:293–301. [19] Kargi F, Uygur A. Nutrient removal performance of a sequencing batch reactor as a function of the sludge age. Enzyme Microb Technol 2002;31:842–7. [20] Kargi F, Uygur A. Nutrient removal performance of a five-step sequencing batch reactor as a function of wastewater composition. Proc Biochem 2003;38:1039–45. [21] Kargi F, Uygur A. Biological treatment of saline wastewater in an aerated percolator unit utilizing halophilic bacteria. Environ Technol 1996;17:325–30. [22] Kargi F, Dincer AR. Biological treatment of saline wastewater by fedbatch operation. J Chem Technol Biotechnol 1997;69:167–72. [23] Kargi F, Dincer AR. Salt inhibition effects in biological treatment of saline wastewater in RBC. J Environ Eng ASCE 1999;25:966–71. [24] Dincer AR, Kargi F. Salt inhibition of nitrification and denitrification in saline wastewater. Environ Technol 1999;20:1147–53. [25] Intrasungkha N, Keller J, Blackall LL. Biological nutrient removal efficiency in treatment of saline wastewater. Water Sci Technol 1999;39:183–90. [26] Abu-ghararah ZH, Sherrard JH. Biological nutrient removal in high salinity wastewater. J Environ Sci Health 1993;28:599–613. [27] Dan NP, Vishvanathan C, Basu B. Comparative evaluation of yeast and bacterial treatment of high salinity wastewater based on biokinetic coefficients. Bioresour Technol 2003;87:51–6. [28] Uygur A, Kargi F. Salt inhibition on biological nutrient removal from saline wastewater in a sequencing batch reactor. Enzyme Microb Technol 2004;34:313–8. [29] American Public Health Association (APHA). Standard methods for the examination of water and wastewater, 17th ed. Washington, DC; 1989.
Specific nutrient removal rates in saline wastewater treatment using sequencing batch reactor Ahmet Uygur * Department of Environmental Engineering, Uludag˘ Universiy, Go¨ru¨kle, Bursa, Turkey Received 21 October 2004; received in revised form 26 January 2005; accepted 12 March 2005
Abstract Effects of salt concentration (0–6%, w/v) on specific nutrient removal rates from saline wastewater in a sequencing batch reactor (SBR) were investigated. The sequencing batch operation consisted of anaerobic, oxic, anoxic and oxic phases with hydraulic residence times (HRT) of 1/3/1/1 h and a settling phase of 3/4 h. Solids retention time (SRT) was kept constant at 10 days in all experiments. Specific nutrient (COD, NH4-N and PO4-P) removal rates decreased with increasing salt concentration due to adverse effects of salt on microorganisms. A salt tolerant organism, Halobacter halobium was added to the activated sludge culture (1/1, v/v) in order to improve the nutrient removal performance of the SBR. Nutrient removal performances of Halobacter-free and Halobacter-added activated sludge cultures were compared for all salt contents tested. Specific rates of nutrient removal obtained with the Halobacter-added culture were higher that those of Halobacter-free activated sludge, especially at high salt contents. # 2005 Elsevier Ltd. All rights reserved. Keywords: Halobacter halobium; Saline wastewater; Nutrient removal; Sequencing batch reactor (SBR)
1. Introduction Biological treatment of saline wastewater usually results in low BOD/COD removal performances because of adverse effects of salt on microbial flora. High salt concentration (>1% salt) causes plasmolysis and loss of activity of the cells. Several researchers have investigated the effects of salinity on bioactivity of microorganisms in biological treatment systems and found that high levels of salinity had adverse effects on treatment performance [1–3]. Matsuo and Hosobora [4] studied the effect of salinity on enhanced biological phosphorus removal and reported that NaCl concentrations above 20 mM (1.17 g l1) restricted the luxury phosphorus uptake resulting in high phosphorus contents in the effluent. However, the system was able to resume its performance after an appropriate acclimation period. Panswad and Anan [5,6] observed an improvement in the carbon and nitrogen removal efficiency in biological nutrient removal from high salinity wastewater when seeded * Tel.: +90 224 4428177; fax: +90 224 4429148. E-mail address: [email protected]. 1359-5113/$ – see front matter # 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.procbio.2005.03.068
with salt-acclimated cultures. Phosphorus-accumulating bacteria were found to be more sensitive to the high salt concentrations than nitrifiers and denitrifiers. Dahl et al. [7] investigated nitrification and denitrification of gas scrubbing liquors which have high chloride content. Maximum rates of nitrification and denitrification were reported as 2 and 3 mgN gVSS1 h1, respectively, at 30 8C and 20 g Cl l1 chloride concentration. Yang et al. [8] investigated biological treatment of brine wastewater generated by ion exchange processes. Because of utilization of entrapped mixed culture, no significant salt inhibition of nitrate removal was observed up to 10 g l1 (1%) salt concentrations. Salt inhibition effects on nitrification of synthetic wastewater were investigated by Kargi and Dincer using an activated sludge unit [9]. Experiments performed with 3% salt content at different sludge ages were used for determination of salt inhibition constants for nitrification. A non-competitive inhibition affecting both the maximum rate and the saturation constants were found to be suitable for salt inhibition of nitrification. Numerous studies in the literature have reported on biological nutrient removal [10–18]. Kargi and Uygur used a
62
A. Uygur / Process Biochemistry 41 (2006) 61–66
five-step SBR for nutrient removal from a synthetic wastewater. Effects of the sludge age (SRT), hydraulic residence time (HRT), feed wastewater composition and carbon sources on nutrient removal performance were investigated. Sludge age of 10 days and a wastewater composition of COD/N/P = 100/3.33/0.69 were determined as optimal values resulting in maximum percent nutrient removal [19,20]. COD removal from saline wastewater was investigated indicating salt inhibition at salt concentrations above 2% [21–24]. However, only a few studies are reported in literature on biological nutrient (COD, N, P) removal from low salinity (salt < 1%) wastewater. Intrasungkha et al. [25] investigated biological nutrient removal from saline wastewater using an SBR with satisfactory nutrient removals at low salt contents (0.03–0.2% NaCl). Abu-ghararah and Sherrard [26] determined that salinity had little effect on nitrogen removal at 0.4% salt content (4 g/l NaCl). However, percent phosphorus removal decreased from 82% with saltfree wastewater to 25 with 0.4% salt. Dan et al. [27] used an osmo-tolerant yeast culture for biological treatment of saline wastewater (2–4.5% NaCl) and compared the treatment performance with that of the bacterial culture. Specific nutrient uptake for the yeast cells was found to be higher than that of the bacterial culture resulting in higher nitrogen and phosphorous removal rates. Effects of high salt contents on the extent of nutrient (COD, N, P) removal in an SBR was investigated for a large range of salt contents above 1% [28]. None of the aforementioned studies on saline wastewater treatment specific nutrient removal rates were investigated for a large range of salt concentrations using an SBR. Therefore, the major objective of this study is to investigate and alleviate the adverse effects of high salt contents on specific nutrient removal rate (SNRR) using an SBR. A fourstep SBR consisting of anaerobic/oxic/anoxic/oxic phases with hydraulic residence times (HRT) of 1/3/1/1 h and a settling phase of 3/4 h was used for COD, ammonium-N, phosphate-P removals from saline wastewater at different salt contents (0–6% salt). The sludge age (SRT) was kept constant at 10 days. In the first phase of the study the adverse effects of salt content on specific nutrient removal rates (SNRR) were investigated by using an activated sludge culture. In the second phase the same experiments were repeated using the salt tolerant bacteria, Halobacter-added (1/1, v/v) activated sludge culture to alleviate the adverse effect of salt on nutrient removal rates. Specific nutrient removal rates for both cultures were compared.
2. Materials and methods 2.1. Experimental set-up A schematic diagram of the experimental set up is shown in Fig. 1. A fermenter (Bioflo IIC, New Brunswick) with 5 l working volume was used as the sequencing batch reactor
Fig. 1. Schematic diagram of the sequencing batch reactor used in experimental studies.
(SBR). The fermenter was microprocessor controlled for aeration, agitation, pH and dissolved oxygen (DO). Aeration was provided by using an air pump and a sparger. Agitation speed was varied between 25 and 300 rpm. DO, pH, and ORP of the nutrient medium were continuously monitored by the relevant probes. 2.2. Wastewater composition Synthetic wastewater used throughout the studies was composed of glucose, sodium acetate, NH4Cl, KH2PO4, MgSO47H2O, NaHCO3 and certain trace minerals such as KCl (20 mg l1), CaCl22H2O (50 mg l1) and FeCl36H20 (50 mg l1). Salt (NaCl) concentration was varied between 0 and 6% (w/v). Typical composition of the synthetic wastewater was COD0 = 1200 50 mg l1, NT = 60 3 mg l1 and PT=18 2 mg l1, resulting in COD/N/P = 100/5/1.5. MgSO4 and NaHCO3 concentrations in the feed were 0.1 and 0.708 g l1, respectively, throughout the studies. The pH of the synthetic wastewater was adjusted to 7.0 at the beginning. 2.3. Organisms A mixed microbial population composed of heterotrophic organisms capable of oxidizing carbonaceous compounds and denitrification; autotrophic nitrifying organisms; anaerobic acid producers and excess phosphate uptaking organisms (Acinetobacter sp.) were used as inoculum culture. Nitrification organisms (Nitrosomonas and Nitrobacter) were obtained from Clemson University, SC, USA. The heterotrophic mixed culture was obtained from Cigli Municipal Wastewater Treatment Plant, Izmir. Anaerobic acid producers were obtained from the first stage (acid formation reactor) of the anaerobic wastewater treatment plant of PAKMAYA Bakers Yeast Company, Izmir, Turkey. The culture was cultivated in laboratory and added to the
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inoculum culture. Acinetobacter calcoaceticus (NRRL-552) obtained from the USDA, National Research Laboratories, Peoria, IL, USA was used for excess phosphate uptake. Heterotrophic organisms were cultivated in the same synthetic nutrient media used in the experiments. The growth media for denitrifying organisms contained 800 mg l1 glucose, 50 mg l1 KH2PO4, 610 mg l1 NaNO3, 20 mg l1 MgSO47H2O and 10 mg l1 Na2S2O3 at pH 7. The growth media used for cultivation of nitrification organisms contained 382 mg l1 NH4Cl, 1510 mg l1 NaHCO3, 93 mg l1 glucose, 60 mg l1 KH2PO4, 20 mg l1 CaCl22H2O and 40 mg l1 MgSO47H2O at pH 7.5. The growth media for Acinetobacter calcoaceticus was composed of 5000 mg l1 glucose, 3000 mg l1 KH2PO4, 1000 mg l1 NH4Cl, 500 mg l1 yeast extract, 3000 mg l1 NaCl and 50 mg l1 MgSO47H2O at pH 7. The growth media for Halobacter halobium was composed of 8 g l1 glucose, 300 mg l1 KH2PO4, 1 g l1 NH4Cl, 250 mg l1 yeast extract, 30 g l1 NaCl (3%, w/v) and 50 mg l1 MgSO47H2O at pH 7.5. These cultures were cultivated in suitable growth media in the laboratory and were used as inocula in the form of a mixed culture. Halobacter halobium (ATCC 43214) was obtained from the American Type of Culture Collection (ATCC), Maryland, USA in lyophilized form and cultivated in salt containing synthetic media in the laboratory. Halobacter culture was mixed with activated sludge culture (1/1, v/v) for the experiments performed with the Halobacter-added activated sludge culture to overcome salt inhibition effects on the specific nutrient uptake rates. The concentration of the mixed microbial culture was approximately 3.14 g l1 at the beginning. 2.4. Experimental procedure The reactor was filled with the synthetic wastewater, inoculated with a mixed culture of microorganisms and was operated batchwise with aeration and mixing for several days to obtain a dense culture to start with. After sedimentation of the organisms, 4 l clear supernatant was removed and 1 l dense culture was completed to 5 l total volume with the defined nutrient medium. Then, anaerobic, oxic, anoxic and oxic operations were applied in sequence. Nitrogen gas was passed through the medium only during anaerobic operation. Agitation speed during anaerobic and anoxic cycles was 25 and 50 rpm, respectively. Anoxic conditions were provided by keeping the DO nearly zero and the ORP between 0 and 100 mV. The medium was aerated and agitated (300 rpm) vigorously during oxic (aerobic) operation. Oxidation reduction potentials (ORP) for the anaerobic, oxic, anoxic and oxic phases were approximately 250, +250, 70 and +250 mV, respectively. Samples were withdrawn from the reactor at the beginning and at the end of each cycle for analysis. At the end of each SBR operation, the organisms were settled for 3/4 h and 4 l of the supernatant wastewater was removed. Settled organisms
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of 1 l volume were used for the next treatment operation with the addition of 4 l fresh nutrient media. A fraction of (1/10) of the culture was removed from the reactor daily before settling to adjust the sludge age to 10 days. Temperature and pH was controlled around T = 25 1 8C and pH 7–7.5. Dissolved oxygen (DO) concentration in oxic (aerobic) phase was kept above 2 mg l1, while the DO levels during anaerobic and anoxic phases were practically zero. A four-step SBR operation consisting of anaerobic/oxic/ anoxic/oxic steps with HRT’s of 1/3/1/1 h was used at all salt concentrations. Every experiment was carried out four times at any salt content tested. Data were collected at the end of each phase for every experiment. The system reached the steady-state after 3 days of operation resulting in almost the same nutrient removal. Since the difference in results was negligible for the last two runs (<3% of the average), no further replicates were conducted. 2.5. Analytical methods Samples were withdrawn from the liquid media at the beginning and at the end of each treatment phase (anaerobic, oxic, anoxic, oxic) and were centrifuged at 6000 rpm for 30 min to remove microorganisms from the liquid medium. Clear supernatants were analyzed for COD, ammonium-N and phosphate-P contents. Standard kit (Merck-Spectroquant) and spectrometric method were used for ammoniumN and phosphate-P analyses. COD, total solids (TS) and total suspended solids (TSS) concentrations were determined by using the Standard Methods [29]. Samples were analyzed in triplicates and average values were reported. DO, pH and ORP measurements were done by using the relevant probes and analyzers. Samples were centrifuged to separate saline water from the biomass and the washed salt-free organisms were used in determining the biomass concentrations. Biomass concentrations were determined by filtering the washed salt-free samples through 0.45 mm membrane filter and drying in an oven at 105 8C to constant weight.
3. Results and discussion 3.1. Specific rate of COD removal Variation of specific removal rates of COD with the salt content is depicted in Fig. 2 for the Halobacter-free and Halobacter-added activated sludge cultures. The specific COD removal rate increased from 34.0 to 42.5 mg COD g biomass1 h1 when the salt content was increased from 0 to 1% for the Halobacter-free activated sludge culture indicating stimulatory effects of low salt contents (<1% salt) on the organisms. Further increases in salt content caused severe decreases in specific COD removal rate resulting in 26.7 and 10.7 mg COD g biomass1 h1. COD removal rates at 3 and 6% salt contents, respectively. Apparently, salt contents above 1% caused cell disintegra-
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A. Uygur / Process Biochemistry 41 (2006) 61–66
Fig. 2. Variation of specific COD removal rate with salt content. (*) Halobacter-free activated sludge culture and (*) halobacter-added activated sludge culture.
tion resulting in lower specific COD removal rates in the activated sludge culture. Specific COD removal rates obtained with the Halobacter-added activated sludge was higher than those of Halobacter-free activated sludge especially at high salt contents. Specific COD removal rate increased from 42.6 to 63.3 mg COD g biomass1 h1 when salt content increased from 0 to 1% in the Halobacter-added activated sludge culture indicating stimulatory effects of low salt contents on the organisms. Further increases in salt content caused decreases in COD removal rates, however to a lesser extent as compared to the Halobacter-free activated sludge. The specific COD removal rate decreased to 44 mg COD g biomass1 h1 at 3% salt and further to 31.0 mg COD g biomass1 h1 at 6% salt content. Apparently, the salt tolerant bacteria, Halobacter halobium was not affected from high salt contents above 1% yielding high COD removal rates as compared to the activated sludge culture. Inclusion of Halobacter culture to the activated sludge was found to be beneficial by providing high COD removal rates. Data presented in Fig. 2 for the specific COD removal rate by the Halobacter-free and Halobacter-added activated sludge culture were correlated with a fourth-order polynomial equation in the following form. Halobacter-free activated sludge: RCOD ¼ 0:55 S4 þ 6:5 S3 24:1 S2 þ 23:75 S þ 36:6 2
ðR ¼ 0:90Þ
3.2. Specific rate of ammonium-N removal Variation of specific removal rate of NH4-N with salt content is depicted in Fig. 3 for the Halobacter-free and Halobacter-added activated sludge cultures. Specific NH4-N removal rate decreased with increasing salt content for both cultures. However, the Halobacter-added culture resulted in higher specific NH4-N removal rates as compared to the Halobacter-free activated sludge at all salt concentrations tested. The specific rate of NH4-N removal decreased from 3.0 to 0.80 mg NH4-N g biomass1 h1 when salt content increased from 0 to 6% for the Halobacter-free activated sludge culture. Halobacter-added activated sludge culture resulted in NH4-N removal rates of 3.04 and 1.45 mg NH4N g biomass1 h1 at 0 and 6% salt contents, respectively. The results indicated that Halobacter addition to the activated sludge culture improved the specific NH4-N removal rates significantly, especially at high salt concentrations. The data presented in Fig. 3 for the specific NH4-N removal rate were correlated with exponential equations in the following forms for both cultures. Halobacter-free activated sludge: RNH4 -N ¼ 3:024 e0:1978S
(1)
RCOD ¼ 0:46 S4 þ 6:3 S3 28:5 S2 þ 42:5 S þ 41:4
ðR2 ¼ 0:93Þ
(3)
Halobacter-added activated sludge: RNH4 -N ¼ 3:152 e0:1204S
Halobacter-added activated sludge:
ðR2 ¼ 0:98Þ
Fig. 3. Variation of specific NH4-N removal rate with salt content. (*) Halobacter-free activated sludge culture and (*) halobacter-added activated sludge culture.
ðR2 ¼ 0:93Þ
(4)
where, RNH4 -N is the specific rate of NH4-N removal (mg NH4-N g biomass1 h1) and (S) is the percent salt concentration. 3.3. Specific rate of PO4-P removal
(2)
where, RCOD is the specific COD removal rate mg COD g biomass1 h1 and (S) is the percent salt concentration.
Variation of specific PO4-P removal rates with the salt concentration are depicted in Fig. 4 for both Halobacter-free and Halobacter-added activated sludge culture. Specific rates of PO4-P removal decreased with increasing salt
A. Uygur / Process Biochemistry 41 (2006) 61–66
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4. Conclusions
Fig. 4. Variation of specific PO4-P removal rate with salt content. (*) Halobacter-free activated sludge culture and (*) halobacter-added activated sludge culture.
content for both cultures. However, the adverse effects of salt were more pronounced with the Halobacter-free activated sludge. The specific rate of PO4-P removal decreased 0.36–0.08 mg PO4-P g biomass1 h1 when salt content increased from 0 to 6% for the Halobacter-free activated sludge culture indicating severe adverse effects of high salt contents on phosphate removal rates. Halobacteradded activated sludge culture resulted in PO4-P removal rates of 0.52 and 0.18 mg PO4-P g biomass1 h1 at 0 and 6% salt contents, respectively. Specific rates of PO4-P removal obtained with Halobacter-added activated sludge culture were higher than those of Halobacter-free activated sludge at all salt contents tested. Plasmolysis of excess phosphate uptaking organisms and release of deposited intracellular phosphate compounds might be responsible for the low the specific PO4-P uptake rates at high salt contents with the Halobacter-free activated sludge. The results indicated clear advantage of using Halobacter in the activated sludge culture in order to obtain high PO4-P removal rates. Halobacter halobium species are known to be incapable of excess phosphate uptake. Therefore, the improvement in specific PO4-P uptake rate at high salt contents is probably because of phosphate assimilation by the Halobacter species for biosynthesis. The specific PO4-P removal rate data presented in Fig. 4 were correlated with exponential equations in the following form for both cultures. Halobacter-free activated sludge: RPO4 -P ¼ 0:282 e0:2278S
ðR2 ¼ 0:79Þ
(5)
Halobacter-added activated sludge: RPO4 -P ¼ 0:448 e0:1780S
ðR2 ¼ 0:89Þ
(6)
where, RPO4 -P is the specific PO4-P removal rate (mg PO4P g biomass1 h1) and (S) is the percent salt concentration.
Specific nutrient removal rates from saline wastewater were investigated using Halobacter-free and Halobacteradded activated sludge cultures in a sequencing batch reactor (SBR). A four-step SBR operation consisting of anaerobic/ oxic/anoxic/oxic steps with 1/3/1/1 h hydraulic residence times (HRT) and a constant sludge age of 10 days was used throughout the studies. Salt content of the synthetic wastewater was varied between 0 and 6%. Specific rates of nutrient removal decreased with increasing salt content for both cultures. However, nutrient removal rates were higher with the Halobacter-added activated sludge culture as compared to the Halobacter-free culture. Low contents of salt (<1%) were found to be stimulatory for the organisms resulting in an improved COD removal rate. Because of the plasmolysis of the activated sludge organisms at high salt contents, specific nutrient removal rates decreased with increasing salt content. Improved specific nutrient removal rates were obtained with the Halobacter-added activated sludge culture especially at salt contents above 1% (w/v), due to the salt tolerant characteristics of Halobacter halobium species. Specific COD, NH4-N and PO4-P removal rates were 10.7 mg COD g biomass1 h1, 0.80 mg NH4-N g biomass1 h1 and 0.08 mg PO4-P g biomass1 h1 for the Halobacterfree activated sludge culture while those values were 31.0 mg COD g biomass1 h1, 1.45 mg NH4-N g 1 1 biomass h and 0.18 mg PO4-P g biomass1 h1 for the Halobacter-added activated sludge. The results indicated clear advantage of inclusion of Halobacter halobium to the activated sludge in order to obtain high nutrient removal rates especially at high salt concentrations.
Acknowledgements The author expresses his gratitude to Prof. Dr. Fikret Kargi for his valuable contribution to the critical review of the paper. This study was supported by the research funds of Uludag University in Bursa and Dokuz Eylul University in Izmir, Turkey.
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