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Denitrification of synthetic concentrated nitrate wastes by aerobic granular sludge under anoxic conditions

Denitrification of synthetic concentrated nitrate wastes by aerobic granular sludge under anoxic conditions
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  See discussions, stats, and author profiles for this publication at: Denitrification of synthetic concentratednitrate wastes by aerobic granular sludge underanoxic conditions  Article   in  Chemosphere · July 2011 DOI: 10.1016/j.chemosphere.2011.06.077 · Source: PubMed CITATIONS 32 READS 50 2 authors:Some of the authors of this publication are also working on these related projects: Biofilm characterization under different treatment exposure of selenate, nitrate and sulfate   ViewprojectBiohydrometallurgy of critical/scarce/precious metals from waste materials   View projectVenkata Nancharaiah YarlagaddaBhabha Atomic Research Centre 59   PUBLICATIONS   846   CITATIONS   SEE PROFILE Vayalam P VenugopalanHomi Bhabha National Institute 146   PUBLICATIONS   1,649   CITATIONS   SEE PROFILE All content following this page was uploaded by Venkata Nancharaiah Yarlagadda on 24 March 2014. The user has requested enhancement of the downloaded file. All in-text references underlined in blue are added to the srcinal documentand are linked to publications on ResearchGate, letting you access and read them immediately.  This article appeared in a journal published by Elsevier. The attachedcopy is furnished to the author for internal non-commercial researchand education use, including for instruction at the authors institutionand sharing with colleagues.Other uses, including reproduction and distribution, or selling orlicensing copies, or posting to personal, institutional or third partywebsites are prohibited.In most cases authors are permitted to post their version of thearticle (e.g. in Word or Tex form) to their personal website orinstitutional repository. Authors requiring further informationregarding Elsevier’s archiving and manuscript policies areencouraged to visit:  Author's personal copy Technical Note Denitrification of synthetic concentrated nitrate wastes by aerobic granularsludge under anoxic conditions Y. Venkata Nancharaiah ⇑ , Vayalam P. Venugopalan Biofouling and Biofilm Processes Section, Water and Steam Chemistry Division, Chemistry Group, Bhabha Atomic Research Centre, Kalpakkam 603 102, Tamil Nadu, India a r t i c l e i n f o  Article history: Received 27 December 2010Received in revised form 20 June 2011Accepted 20 June 2011Available online 13 July 2011 Keywords: Aerobic granular sludgeConcentrated nitrate wastesDenitrificationHigh strength nitrate a b s t r a c t The aim of the present work was to determine the denitrification potential of aerobic granular sludge forconcentrated nitrate wastes. We cultivated mixed microbial granules in a sequencing batch reactor oper-ated at a superficial air velocity of 0.8 cm s  1 . The denitrification experiments were performed underanoxic conditions using serum bottles containing synthetic media with 225–2250 mg L   1 NO 3 –N. Timerequired for complete denitrification varied with the initial nitrate concentration and acetate tonitrate–N mass ratio. Complete denitrification of 2250 mg L   1 NO 3 –N under anoxic conditions wasaccomplished in 120 h. Nitrite accumulation was not significant (<5 mg N L   1 ) at initial NO 3 –N concen-trations below 677 mg L   1 . However, denitrification of higher concentrations of nitrate ( P 900 mg N L   1 )resulted in buildup of nitrite. Nevertheless, nitrite buildups observed in present study were relativelylower compared to that reported in previous studies using flocculent activated sludge. The experimentalresults suggest that acetate-fed aerobic granular sludge can be quickly adapted to treat high strengthnitrate waste and can thus be used as seed biomass for developing high-rate bioreactors for efficienttreatment of concentrated nitrate-bearing wastes.   2011 Elsevier Ltd. All rights reserved. 1. Introduction Nitrate is perhaps the most oxidized contaminant found inground waters worldwide (Lee et al., 2006). Nitrate contaminationof water is a concern because it can cause blue-baby syndrome ininfants (Shrimali and Singh, 2001). In addition, high nitrate levelsin water bodies can cause eutrophication, leading to enhancedgrowth of phytoplankton, often followed by hypoxia and fish-kill.Extensive use of fertilizers in agriculture and improper dischargeof sewage and industrial effluents are the sources for groundwatercontamination with nitrate and nitrite. Physical and chemicaltreatment processes such as reverse osmosis, ion exchange,membrane filtration and electrodialysis are effective for removingnitrate, but are expensive and generate concentrated wastes thatrequire subsequent treatment and disposal (Kapoor and Viraragh-avan, 1997). Biological denitrification is the preferred alternativefor removing nitrate from surface waters (Kapoor and Viraragha-van, 1997) and the most commonly used method for removingnitrate from municipal wastewaters. However, the effluents gener-ated in fertilizer, explosives, pharmaceutical, metal finishing andnuclear industries contain relatively high concentrations(>1000 mg L   1 ) of nitrate (Francis and Mankin, 1977; Glass andSilverstein, 1998, 1999). A few studies have shown that biologicaldenitrification can be used for removing nitrate from concentratedwastes (Francis and Mankin, 1977; Glass and Silverstein, 1998,1999; Foglar et al., 2005; Dhamole et al., 2007; Biradar et al.,2008). Nitrite accumulated during the denitrification of high levelsof nitrate can, in turn, inhibit the whole denitrification process(Francis and Mankin, 1977; Glass et al., 1997; Oh and Silverstein,1999) because most heterotrophic denitrifying bacteria are foundto be inhibited by 200 mg L   1 of nitrite (Glass et al., 1997; Foglaret al., 2005; Chen et al., 2008; 2009; Adav et al., 2010).In biological treatment systems, biomass in the form of granularsludge makes it possible to maintainhigh cell concentrations in thereactor and thereby achieve high conversion rates desired for thetreatment of concentrated effluents. In addition, due to their com-pact structure, granules, like biofilm, can tolerate high concentra-tions of pollutants. In the last decade, aerobic microbial granules(aerobic granularsludge) have attracted immense research interestdue to their potential use in the development of new generationwastewater treatment plants (Di Iaconi et al., 2007; Adav et al.,2008). In these systems, microbial community is grown as compactand dense microbial granules in a sequencing batch reactor (SBR)without using a carrier material (Morgenroth et al., 1997). Previousresearch shows that granular sludge cultivated on acetate can beutilized for toxic pollutant biodegradation through adaptation(Tay et al., 2005) or bioaugmentation (Nancharaiah et al., 2008). Recently, the denitrification of 200 mg L   1 of NO 2 –N by acetate-fed aerobic granules was observed in batch experiments carried 0045-6535/$ - see front matter    2011 Elsevier Ltd. All rights reserved.doi:10.1016/j.chemosphere.2011.06.077 ⇑ Corresponding author. Tel.: +91 44 27480203; fax: +91 44 27480097. E-mail addresses:, (Y.V. Nancharaiah). Chemosphere 85 (2011) 683–688 Contents lists available at ScienceDirect Chemosphere journal homepage:  Author's personal copy out in serum bottles (Adav et al., 2010). It was proposed that biog- ranules can be used to increase the tolerance to high nitrite con-centrations without considerable inhibition on denitrification(Adav et al., 2010). Moreover, presence of nitrate (formed com-monly by nitrification) in SBRs and denitrification of nitrate im-proved the development of aerobic granular sludge at lowersuperficialair velocities (Wanet al., 2009). To our knowledge, therehave been no studies to determine the potential of aerobic micro-bial granules to treat high-strength nitrate wastes.The objective of this study was to cultivate aerobic microbialgranules in SBR and to determine their denitrification capabilityunder anoxic conditions in serum bottles. We carried out the deni-trification experiments by using sodium acetate and sodium ni-trate as electron donor and acceptor, respectively. Denitrificationof synthetic waste containing 225–2250 mg L   1 NO 3 –N was stud-ied in batch experiments using serum bottles. In addition, denitri-fication of 900 mg L   1 NO 3 –N was determined in a fed-batchexperiment. Here, we show that aerobically grown microbial gran-ules cultivatedina SBR can quicklyadapt to denitrifying conditionsand completely denitrify up to 2250 mg L   1 NO 3 –N. 2. Materials and methods  2.1. Cultivation of aerobic microbial granules A 3-L working volume SBR was used for cultivating aerobicmicrobial granules (Nancharaiah et al., 2006a,b). Briefly, the SBR was inoculated with 1 L of wastewater containing activated sludgeflocs (2.8 g volatile suspended solids (VSS) L   1 ) collected from anoperatingmunicipal wastewater treatment plant. SBR was fed withsynthetic wastewater (SWW) prepared in deionized water andconsisting of the following (mM): CH 3 COONa (63), MgSO 4  7H 2 O(3.6), KCl (4.7), NH 4 Cl (35.4), K 2 HPO 4  (4.2) and KH 2 PO 4  (2.1). Traceelements were supplemented by adding 0.1 mL of stock solution to1 L of SWW (Nancharaiah et al., 2008). Air was supplied at a super-ficial air velocity of 0.8 cm s  1 . The SBR was operated at room tem-perature (  28   C) with a volumetric exchange ratio of 66% and 6 hcycle period.  2.2. Denitrification experiments Denitrification efficiency of acetate-fed aerobic granular sludgewas determined in 125 mL serum bottles. SWW for the denitrifica-tion experiments was prepared as described above and containeddifferent concentrations of sodium acetate and sodium nitrate(Table 1). Media and serum bottles were autoclaved separately.Aliquots of 100 mL media were dispensed into 125 mL serum bot-tles containing 4 g (wet weight) of aerobic microbial granules(equivalent to 0.094 ± 0.002 g dry biomass). The serum bottleswere sealed with butyl rubber stoppers, and purged with ultra highpurity nitrogen gas for 10 min. The serum bottles were incubatedat room temperature (  28   C) without mixing. At periodic timeintervals 4 mL of media were removed and used for measuring tur-bidity, pH, nitrate and nitrite. Activated sludge inoculum and aer-obic granular sludge sampled before and after the denitrificationexperiment were used for DNA extraction.  2.3. Microscopy Morphology of the aerobic microbial granules was documentedwith an Olympus DP70 camera connected to a SMZ1000 stereo-zoom microscope (Nikon, Japan). Particle size and circularity of the aerobic microbial granules were determined by using the free-ware  ImageJ   v1.43, as described in Nancharaiah et al. (2006a,b).Microstructure of aerobic microbial granules was determined byconfocal laser scanning microscopy (CLSM) and SEM. For CLSMimaging, the microbial granules were stained with LIVE/DEAD  Bac- Light bacterial viability kit (Molecular Probes, USA) according tothe manufacturer’s instructions. A 200 l L of   Bac  Light stain mixturewas transferred to 1.5 mL Eppendorf tube containing a few micro-bial granules and incubated on an orbital shaker set at 100 rpm.After15 minincubation,the microbialgranules werewashedtwicewith ultrapure water. Stained granules were placed directly on topof a glass cover-slip and imaged using a confocal laser scanningmicroscope TCS SP2 AOBS (Leica Microsystems, Germany)equipped with an inverted microscope (Leica DMIRE2). A63  1.2 NA water immersion objective lens was used for imaging.Argon laser (488 nm line) was used for excitation and emissionwas collected between 500 and 520 nm for SYTO 9 and between600 and 680 nm for propidium iodide. For SEM imaging, the micro-bial granules were fixed overnight with 2.5% glutaraldehyde inphosphate-buffered saline. The fixed granules were subjected todehydration for 3 min each in graded ethanol series (50%, 70%,90% and 100%). The dehydrated microbial granules were sputter-coated with gold–palladium and imaged using a scanning electronmicroscope (Philips XL30 ESEM).  2.4. Analytical procedures At periodic intervals, 4 mL of the media was removed using asyringe and used for measuring turbidity, pH, nitrite and nitrate.Turbidity measurements were made at 600 nm using spectropho-tometer (Shimadzu, Japan) and the pH was determined using aHach pH meter. The samples collected at periodic time intervalswere filtered through a 0.45 l m Millex filter, and nitrate in the fil-trate was analyzed by high pressure liquid chromatography (Dio-nex Ultimate 3000) fitted with a Acclaim OA column. The mobilephase was 0.003 N H 2 SO 4 , pumped at a flow rate of 0.7 mL min  1 .Nitrate was determined using a UV–vis detector set at 210 nm. Ni-trite was determined by N-(1-napthyl)ethylenediamine dihydro-chloride method (APHA, 1998) using spectrophotometer. VSS,sludge volume index (SVI), biomass density and dry weight of bio-massweremeasuredaccordingtoStandardMethods(APHA,1998). 3. Results  3.1. Granule formation The SBR was inoculated with seed sludge which consisted of fluffy, irregular and loose flocs. Microscopic examination of theseed sludge showed dominance of filamentous bacteria. WithSBR operation time, formation of more dense, smooth and nearlycircular microbial granules was evident. After 1 wk of operation,  Table 1 Composition of synthetic media with varying amounts of nitrate and acetate concentrations. Constituents (g L   1 ) NO 3 –N (mg L   1 )225 450 677 900 1130 1355 1580 1806 2258NaNO 3  0.43 0.86 1.30 1.73 2.17 2.59 3.03 3.45 4.32CH 3 COONa (3:1) a 1.33 2.66 4.00 5.31 6.67 7.97 9.33 – –CH 3 COONa (2:1) 0.90 1.79 2.70 3.59 – 5.38 – 7.17 8.97 a Sodium acetate was added to give a C/N molar ratio of 3:1 or 2:1.684  Y.V. Nancharaiah, V.P. Venugopalan/Chemosphere 85 (2011) 683–688  Author's personal copy microbial granules with diameters of 0.1–0.8 mm were observed.After 30 d, the average size of the granules was 1.8 mm. The mor-phology of granular biomass sampled from SBR on day 30 is shownin Fig. 1. The microbial granules exhibited excellent settling abilitywith SVI 10  of 32 mL g  1 and biomassconcentrationof 3.5 g VSS L   1 .Filamentous microorganisms, predominant in the seed sludge,were not observed in the microbial granules. Staining with generalnucleic acid binding fluorophore revealed that the microbial gran-ules consisted of several cell clusters with extensive water chan-nels (Fig. 1b). Majority of the cell clusters consisted of closelyorganized bacterial cells. SEM images showed the presence of rodand cocci shaped cells on the outer surface of aerobic microbialgranules (Supplementary Materials (SM), Fig. SM-1).  3.2. Denitrification at C:N ratio of 3:1 Denitrification potential of aerobically grown microbial gran-ules was determined at two different C/N ratios. Nitrite formationwas not observed in the absence of an electron donor, acetate ormicrobial granules in the media. The denitrification of different ini-tial NO 3  at a C/N ratio of 3:1 is shown in Fig. 2. Formation of notice-able amount of nitrite was observed only after a lag period of 20 h.Nitrite formation was observed, but it was not accumulated duringdenitrification of up to 677 mg L   1 NO 3 –N. Nitrite buildup of lessthan 5 mg N L   1 was observed during denitrification of 225, 450and 677 mg L   1 of NO 3 –N (Fig. 2b). Complete denitrification of 900 mg L   1 NO 3 –N was observed in 48 h. However, relatively highnitrite buildup was observed during denitrification of 900 mg L   1 and higher NO 3 –N (Fig. 2). Nitrite levels rose to 250 mg N L   1 whenthe initial NO 3 –N was increased to 1580 mg L   1 . Nitrite buildupwas determined to be less than 100 mg N L   1 at all other initialconcentrations of nitrate. Denitrification of 1130, 1350 and1580 mg L   1 NO 3 –N was complete and occurred in 48–72 h(Fig. 3). In all the cases, nitrate removal was accompanied by theformation and subsequent disappearance of nitrite. At the end of complete denitrification, nitrate–N concentrations were below1 mg L   1 (data not shown for higher nitrate concentrations).  3.3. Denitrification at C:N ratio of 2:1 Formation, accumulation and disappearance of nitrite duringdenitrification of different initial NO 3 –N at a C/N ratio of 2:1 areshown in Fig. 4. Nitrite accumulation and the time required forcomplete denitrification varied with the initial nitrate concentra-tion. Formation of noticeable amount of nitrite was observed onlyafter a lag period of 24 h. Nitrite levels rose steadily and subse-quently decreased to less than 1 mg N L   1 , showing complete deni-trification. Nitrite accumulation was lower than 200 mg N L   1 during denitrification of up to 900 mg L   1 NO 3 –N. Nitrite levelsrose to approximately 900 mg N L   1 when the initial NO 3 –N wasincreased to 2250 mg L   1 . Denitrification of 900, 1350, 1800 and2250 mg L   1 of NO 3 –N was complete and occurred in 60, 72, 96and 120 h respectively. At the end of complete denitrification,the nitrate–N was found to be below 1 mg L   1 .  3.4. Denitrification in a fed-batch experiment  Denitrification pattern during three cycles in a fed-batch exper-iment is shown in Fig. 5. A lag period in denitrification of nitrate Fig. 1.  Morphology and microstructure of aerobic microbial granules. (A) Photo-micrograph of aerobic microbial granules sampled on day 30 of SBR operation. Scalebar = 1 mm. (B) Confocal microscopic image of a single aerobic microbial granuleused in denitrification experiments. B consisted of 28 confocal  xy -slices obtained ata  z  -interval of 1 l m. Live cells are green while membrane compromised cells appearred.  XY   = 240 l m,  Z   = 40 l m. (For interpretation of the references to colour in thisfigure legend, the reader is referred to the web version of this article.) Fig. 2.  Nitrate (A) and nitrite (B) profile during denitrification of 225, 450, 677 and900 mg L   1 of NO 3 –N by aerobic microbial granules. Experiments were performedwith a C/N molar ratio of 3:1. Data points in (B) are mean of three independentexperiments. Only one set of samples were analyzed for nitrate. Error barsrepresent ±1SD (not clearly visible in some of the data points). Y.V. Nancharaiah, V.P. Venugopalan/Chemosphere 85 (2011) 683–688  685
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