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Bioresource Technology 128 (2013) 273–280
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Bioresource Technology
journal homepage: www.elsevier .com/locate /bior tech
Nitrophenol removal by simultaneous nitrification denitrification (SND)using T. pantotropha in sequencing batch reactors (SBR)
Pradnya Kulkarni ⇑Center for Environmental Science and Engineering, Indian Institute of Technology Bombay, Powai, Mumbai 400076, India
h i g h l i g h t s
" Complete detoxification anddegradation of nitrophenols wasachieved via SND in SBR.
" SND was accomplished with a singlesludge biomass containingThiosphaera pantotropha.
" T. pantotropha can provide anattractive choice for SBR due to highpotential of SND.
" Total nitrogen removal was achievedalong with nitrophenol removal.
" SND based SBR offers operationalsimplicity and flexibility along withhigh economy.
0960-8524/$ - see front matter � 2012 Elsevier Ltd. Ahttp://dx.doi.org/10.1016/j.biortech.2012.10.054
Abbreviations: SND, simultaneous nitrificationsequencing batch reactors; 4-NP, 4-nitrophenol;2,4,6-TNP, 2,4,6-trinitrophenol; HRT, hydraulic reteoxygen demand; NO2-N, nitrite nitrogen; NO3-N, nperformance liquid chromatogram; DO, dissolved oxy⇑ Tel.: +91 11 26591391.
E-mail addresses: [email protected], prad
g r a p h i c a l a b s t r a c t
Reactor R1 4 NP SBR
Reactor R2 2,4 DNP SBR
Reactor R3 2,4,6 TNP SBR
Variab
le flow
W
orkin
g
Vo
lum
e 5L
4 NP SBR 2,4 DNP SBR 2,4,6 TNP SBRBackground Studies (R)
Control SBR
COD Removal 4-NP = 99%
2,4-DNP = 97-98%2,4,6-TNP = 97-98%
Nitrite - Nitrate Balance Nitrite non-detectable and
Nitrate 15-30 mg/l for 4NP, 2,4-DNP and 2,4,6-
TNP
NP Removal 4-NP = 98%
2,4-DNP = 83 to 84%. 2,4,6-TNP = 83 to 84%.
Nitrophenols - Sole Source of Nitrogen
SND Simultaneous
Nitrification and Denitrification
Single Sludge Biomass Acclimation Studies
a r t i c l e i n f o
Article history:Received 9 August 2012Received in revised form 11 October 2012Accepted 12 October 2012Available online 23 October 2012
Keywords:NitrophenolsThiosphaera pantotrophaHeterotrophic nitrificationAerobic denitrificationSBR
a b s t r a c t
Nitrophenol removal was assessed using four identical lab scale sequencing batch reactors R (backgroundcontrol), R1 (4-nitrophenol i.e. 4-NP), R2 (2,4-dinitrophenol i.e. 2,4-DNP), and R3 (2,4,6-trinitrophenol i.e.2,4,6-TNP). In the present study, the SND based SBR system was used to carry out total nitrogen removalat reduced aeration (DO = 2 mg/L) using a specifically designed single sludge biomass containing Thiosph-aera pantotropha. The concentration of each of the nitrophenols was gradually increased from 2.5 to200 mg/L during acclimation. The nitrophenols were used as the sole source of nitrogen during study.A synthetic feed was designed to direct SND in the bioreactors. It was observed that overall removalfor 4-NP was 98% and for 2,4-DNP and 2,4,6 TNP, removals varied between 83% and 84%. The CODremoval for 4-NP was 99% and for 2,4-DNP and 2,4,6-TNP was 97–98% during acclimation. Total nitrogenand nitrophenol removals were achieved via SND.
� 2012 Elsevier Ltd. All rights reserved.
1. Introduction
Nitroaromatics are important synthons due to the versatilechemistry of the nitro group. Nitro substituted aromatic
ll rights reserved.
and denitrification; SBR,2,4-DNP, 2,4-dinitrophenol;ntion time; COD, chemicalitrate nitrogen; HPLC, highgen.
compounds such as nitrophenols are important building blocksand intermediates for large-scale production of pesticides, phar-maceuticals, plastics, azo dyes, pigments, wood preservatives, rub-ber chemicals and explosives (Bhatti et al., 2002; Karim and Gupta,2006; Kulkarni, 2012). As these compounds are frequently used inindustrial, agriculture and defense purposes, they are found intothe effluents of these sources, soil and subsurface environment(Feng et al., 2011; Leven et al., 2012). During the production ofnitrobenzene from benzene, TNP (2,4,6-trinitrophenol) and DNP(2,4-dinitrophenol) are generated as off-stream chemicals, whichaccumulate in industrial wastewaters. Hao et al. (1993) reported
274 P. Kulkarni / Bioresource Technology 128 (2013) 273–280
typical composition of nitrotuluene manufacturing red waster con-taining (4-NP) 4-nitrophenol, (2,4-DNP) 2,4-dinitrophenol andother nitrophenolic mixtures together. Nitro aromatics form animportant group of recalcitrant and eco toxic xenobiotics exclu-sively generated from anthropogenic source (Spain, 1995; Sharmaet al., 2012; She et al., 2012).
Sequencing batch reactor (SBR) offers promising alternative tocontinuous Completely Stirred Tank Reactors as it allows both dy-namic conditions and optimal substrate concentration with a largespectrum of operating conditions (easily obtainable on time scale)and high operation flexibility (Tomei et al., 2004). In the specificcase of phenolic wastewater treatment, the suitability of SBR hasbeen proven (Tomei et al., 2003). The anaerobic–aerobic SBR re-quires less energy input which leads to lesser aeration require-ments. However, inappropriate control of anaerobic and aerobicresidence time in SBR system leads to difficulties in controllingthe anaerobic–aerobic microbial consortia. A high level of sophisti-cation is required in timing units, controller software and censors,while automated switches and valves are required to achieve effi-cient organic removal in conventional anaerobic–aerobic SBR sys-tem (Chan et al., 2009).
The traditional or conventional biological process used inwastewater treatment to achieve nitrogen removal involves sepa-rate aerobic and anaerobic phases that are generally carried outin separate bioreactors or by using different aeration intervals(Metcalf and Eddy, 2003). SND is simultaneous heterotrophic nitri-fication and aerobic denitrification where both nitrification anddenitrification are achieved simultaneously under reduced aera-tion and hence the controlling of aerobic and anaerobic consortiais not required. A single sludge biomass or consortium can carryout both nitrification and denitrification simultaneously withoutoxic–anoxic phase change, offering a simpler control. Some hetero-trophic bacteria, e.g. Alcaligenes faecalis (van Niel et al., 1992) andThiosphaera pantotropha (Robertson and Kuenen, 1988), are capa-ble of performing SND by using organic substrates as sources ofcarbon and energy to convert ammonium aerobically into nitrogengas (Ferguson, 1994; Robertson et al., 1988; Stouthamer et al.,1997). T. pantotropha offers great potential for SND (Robertsonet al., 1988) which is highly active under fully aerobic conditions.
The nitrification step is often rate limiting in the nitrogenouswastewater treatment due to slow growth rate of nitrifiers (Gupta,1997). In the view of its higher growth rate and ability to convertammonia to nitrogen gas, the use of T. pantotropha can providean attractive option to wastewater treatment for simultaneouslyremoving two pollutants viz. carbon and nitrogen. In the presentstudy, the feasibility of a single sludge biomass system containingT. pantotropha was evaluated to achieve biodegradation of nitro-phenols (mono, di and tri) in SND based SBR.
2. Methods
2.1. Experimental set up
The batch experiments were conducted in four identical SBRsnamely R, R1, R2 and R3, having a working volume of 5 L. The reac-tor R was kept as background control whereas R1, R2 and R3 werefed with 4-NP (4-nitrophenol), 2,4-DNP (2,4-dinitrophenol) and2,4,6-TNP (2,4,6-trinitrophenol) respectively. All the reactors weremaintained at room temperature (27 ± 4 �C) throughout the study.The reactors were made up of plastic material with a total capacityof 10 L. Air was supplied by a variable flow compressor throughdiffuser. The conventional aerobic–anoxic phase changing wasnot done in these SBR reactors due to specially designed singlesludge containing T. pantotropha. The experimentation was carriedout at reduced aeration (DO = around 2 mg/L) owing to the
inherited property of the single sludge system for SND. For denitri-fication, anoxic mode of operation was not used in the react phase.Each SBR cycle included, a fill phase of 30 min, a react phase withaerobic mode (47 h) with anoxic mode-nil, a settle phase of 20 min,a draw/decant phase of 10 min and wastage of 1–2 min. Each SBRcycle lasted 2 d during acclimation studies. After settling, 2.5 L ofthe treated effluent was drawn and around 2.5 L of sludge wasmaintained in the reactor for the next cycle, which gives 4 d ofhydraulic retention time. Synthetic feed (2.5 L) was fed to the reac-tor during each cycle i.e. 50% volumetric exchange ratio. MLSS waskept 3000–4000 mg/L. SRT was 20 d after stabilization of biomasswith peptone and yeast extract. F/M: 0.7 mg COD/mg VSS/day.Decanting volume/Total volume was 0.5.
2.2. Microscopic examination of the biomass and T. pantotropha inbioreactors
The development and the microbial populations of the reactorbiomass were studied using a Zeiss microscope with an imageanalysis software using oil immersion, phase contrast and darkfield modes up to 10�, 40� and 100�magnifications. ESEM exam-ination of biomass was carried out to study structure and morpho-logical features of the biomass. During the examination, biomasssamples from the reactors and plates were taken directly withoutprocessing and fixing, dehydrated using solvents and put on thestab of ESEM.
The presence of T. pantotropha was routinely checked usingZeiss optical microscope with image analysis software under 40�magnification. Gram staining and motility was routinely checkedfrom all four reactors. T. pantotropha showed Gram negative cocc-oidal morphology and non-motile motility.
2.3. Composition of the inocula
The mixed single sludge was prepared specifically with adairy sludge (aerobic) and cow dung along with a biomass ofT. pantotropha. The activated sludge (5 L) was obtained from theMahanada Dairy effluent plant at Goregaon, Mumbai. The cultureT. pantotropha ATCC No. 35512TM was obtained from theDr. Robertson Laboratory, University of Delft, The Netherlands.
2.4. Isolation, identification and enumeration of T. pantotropha frombioreactors
The tentative concentration of T. pantotropha in the bioreactorswas determined by plate counting methods. Untreated mixed li-quor samples from bioreactors were serially diluted (10�8 to10�10), homogenized, and then directly inoculated onto solid med-ia. The techniques employed for the inoculation of agar plates werethe streak-plate and spread-plate techniques. Plates were incu-bated at 30 �C for 4–5 d followed by isolation of well-defined colo-nies onto fresh agar plates. For total bacterial count nutrient agar(NA) was used and for taking T. pantotropha count, selective med-ium with 300 mg/L MgSO4 was used as described by Kshirsagar(1995). The biochemical tests that were used for T. pantotrophaselection from other bacterial isolates include:
2.4.1. Carbohydrate utilization (glucose, maltose, fructose)Test is carried out with peptone broth 4% containing 5% of each
sugar viz glucose, maltose and fructose. Phenol red was added asindicator. Red to yellow colour formation due to pH change inthe culture (T. pantotropha) inoculated tube is indication of positivetest due to acid and gas production. Broth tubes were incubated at37 �C for 24 h. Control was maintained without inoculation ofculture.
P. Kulkarni / Bioresource Technology 128 (2013) 273–280 275
2.4.2. Gelatin hydrolysisGelatin was used as substrate to study proteolytic activity of
microorganisms. Tubes with nutrient gelatin were inoculated withT. pantotropha. Control tube was maintained without inoculation ofthe culture in the Gelatin. Tubes were incubated at 37 �C for 24–48 h and then freezed. The positive test is indicated by presenceof fluid in gelatin tube even after freezing. Microorganisms pos-sessing gelatinase enzyme hydrolyze gelatin giving liquid appear-ance whereas solidification of gelatin indicates negative test.
2.4.3. Indole productionTryptone broth 1% was used. Broth was inoculated with T. pan-
totropha culture. Control was kept without inoculation of culture.Tubes were incubated at 37 �C for 24–48 h presence of indolewas checked with Kovacs reagent. Alcohol layer was separatedfrom aqueous layer upon standing. Reddening of alcohol layerusing Kovacs reagent indicates positive test.
T. pantotropha showed positive test for carbohydrate utilization(glucose, maltose and fructose) and negative test for gelatin hydro-lysis an indole production. It was found that the T. pantotropha wasalways a dominant species of microbial population in all the reac-tors. It constituted about 60–70% of total microbial population inall four bioreactors containing 4-nitrophenol, 2,4-dinitrophenol,2,4,6-trinitrophenol and background control reactor. The othermajor populations which were noted in 4-nitrophenol and 2,4-dinitrophenol reactor were slightly pinkish opaque colony showingchains of gram-positive bacteria and medium translucent colonyshowing presence of gram negative short rods. T. pantotropha gavesmall whitish translucent colonies on plate with gram-negativecoccoidal morphology in Gram staining.
2.5. Preparation of the single sludge
The single sludge biomass was prepared with specific microbialcomposition using simple microbiological techniques. Seed culture(T. pantotropha) and sludge [Dairy sludge + cow dung (1:1)] weremixed in equal proportion, during the start up of bioreactors. Theseed culture (T. pantotropha) was key parameter to carry out SNDin the bioreactors. The proportion of T. pantotropha was specificallymaintained in the bioreactors by adjusting key feed componentMgSO4 to 300 mg/L (Kshirsagar, 1995). The single sludge contained60–70% population of T. pantotropha which was routinely con-firmed with biochemical and plate count methods. The aerobicsludge (dairy sludge) was added to provide various microorgan-isms other than bacteria e.g. ciliates, flagellates, rotifers to facilitatefood chains and food webs in the single sludge (Gupta et al., 1992;Kshirsagar et al., 1994). The cow dung was added to enhance set-tling properties of the single biomass (SVI 45–65 mL g�1). TheMLVSS (Mixed Liquor Volatile Suspended Solids) to MLSS (MixedLiquor Suspended Solids) ratio was maintained around 0.75–0.8in all four bioreactors.
2.6. Analytical procedures
The analytical procedures for all tests were according to thoseoutlined in the Standard methods (APHA, 1998). Daily measure-ments were taken for the influent and effluent pH, COD, NO3
�,NO2
�. The nitrophenol estimation was done with HPLC. The sludgesamples were analyzed for suspended solids (SS) and volatile sus-pended solids (VSS) on biweekly basis. The pH of sample was mea-sured within 5 min of withdrawal in order to minimize pH changesusing a pH meter (Control Dynamics, India) with glass electrode.The nitrophenols were analyzed by injecting 50 lL filtered liquidsamples to HPLC (Shimadzu, LC_6A, Japan) equipped with UV–Visdetector (SPD-6A) and C18 reverse-phase column (250 mm �4.6 mm, 5 n ll ODS, Hypersil, UK). The detection wavelength used
was 280 nm for 4-NP, 2,4-DNP and 2,4,6-TNP. Mobile phase was1:1 deionised water and HPLC grade methanol at flow rate of1 mL min�1. Minimum detection limit was 0.5 mg/L for eachnitrophenol.
3. Results and discussion
Nitrophenol concentration was slowly increased from 2.5 to200 mg/L in small increments in each nitrophenol bioreactor.Acclimation was carried in two phases. In first phase nitrophenolswere degraded by co-metabolism (Perry, 1979) where sodium ace-tate, acetone, peptone, yeast extract and nitrophenols were used ascarbon source (total COD equal to 1000 mg/L) and nitrophenols,peptone and yeast extract were used as nitrogen source. However,in the second phase, nitrophenols were used as the sole source ofnitrogen. Other nitrogen sources such as yeast extract and peptonewere discontinued. This helped in rapid utilization of nitrophenolsalong with rapid development of the single sludge biomass. The fi-nal MLSS maintained was 3000–4000 mg/L in all bioreactors. Forthe background control, sodium acetate and KNO2 were used ascarbon and nitrogen source throughout the study. In the beginning,owing to continuous decrease in the VSS concentration, yeast ex-tract and peptone were added in the bioreactors along with sodiumacetate maintaining a total COD of 1000 mg/L to increase VSS.Within a period of 1 month, the VSS increased from 1000 to2500 mg/L in all four reactors.
3.1. Background studies
A SBR rector (R) was prepared with a single sludge containing T.pantotropha (with the proportion same as that of the nitrophenolbioreactors) using simple carbon and nitrogen source. This SBRreactor (R) was used to compare the toxic effect of nitrophenolson the biomass composition. The basic focus of the present workwas to develop a single sludge with specific composition of micro-organisms to facilitate nitrophenol and complete nitrogen removal.The biomass control (R) was maintained to study deformation inthe single sludge due to nitrophenol addition. As shown inFig. 1(a) and (b), the background control reactor (R) showed goodperformance during the acclimation studies. Sodium acetate wasused as a carbon source and KNO2 was used as a nitrogen sourcefor the background control bioreactor. The reactor was started witha COD of 1000 mg/L and starting KNO2 concentration of 50 mg/L.The concentrations were increased in stepwise manner as nitro-phenol concentrations were increased in nitrophenol reactors.Nitrogen source i.e. KNO2 was increased stepwise from 50, 100,150 and 200 mg/L. Stepwise increase in COD from initial 1000 to4500 mg/L is shown in Fig. 1(a) during acclimation studies.
At each increment of simultaneous COD and KNO2 concentra-tions, the background control reactor was operated for 2 weekssimilar to nitrophenol acclimation in nitrophenol bioreactors. Dur-ing the acclimation studies, COD removal was 99% for the back-ground-control bioreactor indicating the good performance. TheCOD, which was as high as 4500 mg/L, was completely acceptedand removed by the background control bioreactor {Fig. 1(a)}.NO2
� was completely assimilated to NO3� indicating complete
nitrification. Similarly, low levels of NO3� (10–20 mg/L) indicate
complete denitrification in the background control bioreactor(Fig. 1b). The performance of the background control bioreactorindicates successful heterotrophic nitrification and aerobic denitri-fication with mixed single sludge containing T. pantotropha with-out altering the oxic/anoxic phases. Total carbon and nitrogenremovals were achieved with the specially designed mixed singlesludge biomass. It took 70 days for the single sludge biomassto achieve an acclimation as high as 200 mg/L for each of 4-NP,
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Fig. 1. Performance of background control reactor during acclimation: (a) CODremoval and (b) nitrite and nitrate release.
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Fig. 2. 4-Nitrophenol removal during acclimation studies: (a) COD removal and 4-nitrophenol removal and (b) nitrite and nitrate release.
276 P. Kulkarni / Bioresource Technology 128 (2013) 273–280
2,4-DNP and 2,4,6-TNP. The background control bioreactor wasacclimated with 200 mg/L of KNO2.
3.2. Acclimation studies for nitrophenols
The acclimation studies for 4-nitrophenol, 2,4-dinitrophenoland 2,4,6-trinitrophenol showed almost similar profile. In thebeginning, owing to the toxicity of nitrophenols, the MLVSS levelsin the reactors decreased. In order to increase the MLVSS levels,peptone and yeast extracts were added in the nitrophenol reactorsalong with sodium acetate leading to a total COD of 1000 mg/L. Asharp increase in the VSS concentration along with rapid acclima-tion was obtained due to the addition of peptone and yeast extract.The initial increase in MLSS and MLVSS till 50 mg/L of nitrophenolswas contributed by the peptone and yeast extract. However, whenacclimation was achieved till 50 mg/L of nitrophenols, yeast ex-tracts and peptone were discontinued and the excess biomasswas removed and maintained around 3000–4000 mg/L in all biore-actors during further increments of nitrophenols. The major pur-pose of addition of peptone and yeast extract was to stabilize thebiomass and allow it to withstand toxicity of nitrophenols whichwas critical during the initial phase of acclimation when biomasswas damaged and increase in its growth was required. Once thiswas achieved, peptone and yeast extracts were discontinued andthe excess biomass was removed.
The performance of 4-nitrophenol bioreactor during acclima-tion studies is shown in Fig. 2. From Fig. 2(a) and (b), it can be seenthat COD and 4-nitrophenol removals were 99% and 98% respec-tively during acclimation and start up. 4-Nitrophenol concentra-tion was increased in a stepwise manner. The stepwise increasein the COD to a level as high as 4500 mg/L was easily acceptedand completely removed by the single sludge containing T. pantot-
ropha (Fig. 2a). Almost 99% COD removal was obtained whichmeans the COD was either used for VSS production or oxidizedto CO2.
Similarly, a stepwise increase in 4-nitrophenol concentration asshown in Fig. 2(a) was removed successfully by the single sludge.The bioreactor could remove 4-nitrophenol concentrations as highas 200 mg/L and at the same time, nitrite released via 4-nitrophe-nol breakage was simultaneously nitrified to nitrate. Hence theoverall nitrite levels were found to be very low or almost negligible(Fig. 2b). Nitrite initially appeared in effluent but as single sludgebiomass got acclimated to 4-nitrophenol; it became almost negligi-ble. This was due to complete nitrification of nitrite released fromthe breakage of 4-nitrophenol by heterotrophic nitrification to ni-trate, which was in the range of 20–30 mg/L, indicating simulta-neous aerobic denitrification by T. pantotropha to N2. This is anindication of total nitrogen removal occurring in 4 nitrophenol bio-reactor along with total removal of carbon or COD.
For the 2,4-dinitrophenol bioreactor the pattern of biodegrada-tion of 2,4-dinitrophenol, COD, and the release of NO3
� and NO2�
was almost similar to the pattern observed in the 4-nitrophenoldegrading bioreactor. A stepwise increase in the COD to a level(Fig. 3a) as high as 4500 mg/L was easily accepted and completelyremoved by the single sludge containing T. pantotropha for 2,4-dinitrophenol. COD removal of 97–98% was obtained which meansthe COD given was either used for VSS production or was oxidizedto CO2. Similarly, a stepwise increase in 2,4-dinitrophenol concen-tration as shown in Fig. 3(a) was removed successfully to the ex-tent of 83–84% by the mixed single sludge. The only difference in2,4-dinitrophenol removal was that a small fraction of the endmetabolite was detected in the effluent using HPLC. However, itwas found to be a slow degrading fraction as it never accumulatedin the bioreactor. A 2,4-dinitrophenol concentration as high as200 mg/L was successfully removed by the mixed single sludgeand the nitrite released via 2,4-dinitrophenol breakage was
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Fig. 4. 2,4,6-Trinitrophenol removal during acclimation studies: (a) COD removaland 2,4,6-trinitrophenol removal and (b) nitrite and nitrate release.
P. Kulkarni / Bioresource Technology 128 (2013) 273–280 277
simultaneously nitrified to nitrate. Hence, the overall nitrite levelswere found to be very low or almost negligible (Fig. 3b).
The NO2� and NO3
� release pattern for 2,4-dinitrophenol, indi-cated that complete nitrification and denitrification occurredsimultaneously in 2,4-dinitrophenol bioreactor. As seen inFig. 3(b), nitrite concentration was found to be almost negligible,which indicates complete nitrification of NO2
� to NO3� via hetero-
trophic nitrification. Similarly, low levels of NO3� up to 30 mg/L
indicate its simultaneous denitrification to N2. From the resultsshown in Fig. 3(a,b), it is clear that total nitrogen removal tookplace in 2,4-dinitrophenol SBR. Not only phenol ring from 2,4-dini-trophenol, but NO2
� that was released after breakage was alsocompletely removed. This shows remarkable property of the singlesludge containing T. pantotropha for simultaneous removal of thetwo pollutants viz. carbon and nitrogen.
Even for the bioreactor containing 2,4,6-trinitrophenol, the pat-tern of biodegradation of 2,4,6-trinitrophenol, COD removal, re-lease of NO3
� and NO2� was similar to that observed in the other
two nitrophenol degrading bioreactors. A stepwise increase inthe COD (Fig. 4a) to a level as high as 4500 mg/L was easily ac-cepted and was completely removed by the mixed single sludgecontaining T. pantotropha. A COD removal of 97–98% was obtained,which means COD given was either used for MLVSS production oroxidized to CO2.
Similarly a stepwise increase in 2,4,6-trinitrophenol concentra-tion as shown in Fig. 4(a) was removed (83–84%) successfully, bythe mixed single sludge. During 2,4,6-trinitrophenol removal,similar to 2,4-dinitrophenol, a small fraction of the metabolitewas detected in the effluent using HPLC. However, this was foundto be slow degrading fraction, as it never accumulated in the biore-actor. The 2,4,6-trinitrophenol concentration was increased in astepwise manner till a level as high as 200 mg/L, was achievedFig. 4(a) and was successfully removed by the mixed singlesludge. Similarly, NO2
� released via 2,4,6-trinitrophenol breakage
was simultaneously nitrified to NO3� (heterotrophic nitrification).
Hence, the overall NO2� levels were found to be very low or almost
negligible (Fig. 4b). Low levels of NO3� (15–30 mg/L) indicate its
simultaneous denitrification to N2. As discussed previously,NO2
� and NO3� release pattern for 2,4,6-trinitrophenol, showed
complete nitrification and denitrification (SND) which occurredsimultaneously in the SBR.
The comparison of trends in 4-NP, 2,4-DNP and 2,4,6-TNP bio-degradation in the SBR reveals almost similar pattern for CODand nitrophenol removals as well as NO2
� and NO3� release. Suc-
cessful nitrophenol removal was achieved via SND along with com-plete nitrogen and carbon removal using the single sludge with T.pantotropha. As discussed previously, NO2
� and NO3� release pat-
tern for 2,4,6-trinitrophenol, showed complete nitrification anddenitrification (SND) which occurred simultaneously in the SBR.
The loss of nitrogen can be easily found out by mass balanceanalysis of N in influent and effluent. Similarly, in present studythe loss of nitrogen was calculated in terms of nitrite levels inthe influent and effluent samples from bioreactors. Nitrophenolswould give nitrite (NO2
�) and phenol ring upon degradation inthe bioreactors. In present study only partial nitrification is in-volved from nitrite to nitrate and not ammonia to nitrate, hence ni-trite balance is critical and would give exact nitrogen loss ratherthan measuring N in influent, N in effluent. During present studynitrification (nitrite released on breaking of nitrophenols) anddenitrification (nitrate produced) was continuously monitored.The influent nitrite levels were calculated by molecular propertiesof each nitrophenol.
SND using a single sludge biomass containing T. pantotrophawas found to be a successful approach for nitrogen removal inSBR. In conventional nitrification–denitrification system, two dif-ferent consortia or populations are involved to achieve aerobicnitrification followed by anoxic denitrification (combined process).Controlling two different populations together is difficult. Thereis always a possibility that denitrifiers could predominate over
278 P. Kulkarni / Bioresource Technology 128 (2013) 273–280
nitrifiers and consume carbon sources rapidly due to their rapidgrowth rate. This would result in poor nitrification activity. How-ever, in SND as only single consortium is involved for both nitrifi-cation and denitrification, oxic–anoxic phase change or any type ofbiochemical change is not needed for the combined process.
In wastewater treatment, the nitrification step is often cumber-some. The low growth rate of nitrifying bacteria and the relativelypoor capacity of activated sludge units to retain nitrifying biomassrequire large settlers. Slow-growing nitrifiers mainly determinethe residence time in the nitrification unit. The most commonproblem is the apparition of wash out (Campos et al., 2007). Forthese reasons the activated sludge units cannot treat high nitrogenloading rates. In view of its higher growth rate and ability to con-vert ammonia to nitrogen gas, use of T. pantotropha can provide anattractive alternative to wastewater treatment for simultaneouslyremoving two pollutants viz. carbon and nitrogen (Gupta, 1997).The metabolic capacity of this bacterium throws open interestingpossibilities for its applications in wastewater treatment. It is ableto denitrify using reduced sulfur compounds, hydrogen or a widerange of organic compounds as electron donors (Kuenen et al.,1992). It can also nitrify ammonia heterotrophically to nitrite,and reduce nitrate or nitrite to molecular nitrogen gas irrespectiveof the ambient dissolved oxygen concentration (Gupta, 1997). Itdoes not require prior carbon removal step needed before nitrifica-tion, external carbon source is not needed for denitrification, lesserbuffer quantity needed as alkalinity generated during denitrifica-tion can partly compensate for the alkalinity destroyed in nitrifica-tion, acclimation problems are minimized as faced in a single stageoxic–anoxic system. Thus the single sludge system containing T.pantotropha can offer many advantages over conventional aero-bic–anoxic biomass system of SBR or any other reactor systemfor nitrogen removal.
The heart of the SBR is its Operation and Maintenance that in-clude operational controls, automatic valves, automatic timingunits, automated operation systems, etc. that help in aerobic/an-oxic/anaerobic sequencing. A high level of sophistication is re-quired in timing units, controller software and censors, whileautomated switches and valves are required to achieve efficient or-ganic removal in conventional aerobic-anoxic SBR system (Chanet al., 2009). These systems may require more maintenance thana conventional activated sludge system. An increased level ofsophistication usually equates to more items that can fail or re-quire maintenance. The level of sophistication may be very ad-vanced in larger SBR wastewater treatment plants requiring ahigher level of maintenance. Due to high Operation & maintenancerequirements (O & M cost) for operational and control systems, SBRmay be more cost intensive than conventional activated sludgesystem (USEPA, 1999). SND based SBR containing T. pantotrophaas proposed in present study, can carry out nitrification and deni-trification of nitrogenous toxic compounds without altering oxic–anoxic phases in the SBR. The SND process represents a significantadvantage over the conventional separated nitrification and deni-trification processes in SBR with added merits of T. pantotrophain simultaneous carbon and nitrogen removal.
3.3. Selective feed
The selective feed was designed to provide the desired sequenceof biochemical reactions in SBRs. The purpose was to maintain asignificant population of T. pantotropha in the bioreactors and al-low the biodegradation pathways to proceed in favor of heterotro-phic nitrification and aerobic denitrification. Initially, T.pantotropha was grown in a media as prescribed by Robertsonet al. (1988) and adapted to nitrophenols. The combined biomassof dairy and cow dung (1:1) was separately acclimatized to nitro-phenols in the media prescribed by Tomei et al. (2004) with the
only difference that NH4SO4 was replaced by KNO2. The media pre-scribed by Tomei et al. (2004) was continued during start up of theSBRs. The C:N:P ratio was maintained as 100:5:1. However, thismedia was further discontinued as alkalinity started increasing inall the reactors (the pH was more than 9). This showed the neces-sity to increase buffer concentration. Media prescribed by Bhattiet al. (2002) was used with high concentration of K2HPO4
(800 mg/L) and KH2PO4 (200 mg/L). This media provided a pH of7 to 7.5, but was non selective for autotrophic and heterotrophicnitrifiers and denitrifiers. KNO2 was used as a nitrogen source inthe media instead of NH4SO4.
This media was then modified for the supportive nitrogensource (KNO2) to make it more selective for heterotrophic nitrifi-ers. To achieve this, KNO2 was removed from media composition.The idea behind this was to prevent co-metabolic mode ofnitrophenol degradation and use nitrophenol as the sole sourceof nitrogen. This provided selective pressure for microorganismsto degrade nitrophenols, as there was no other source of nitrogenthat was available in the media. Removal of inorganic nitrogensource from the media composition prevented the population ofautotrophic nitrifiers and denitrifiers. However, within theheterotrophic nitrifier population to increase T. pantotropha con-centration, media was further modified for MgSO4 concentrationfrom 100 to 300 mg/L to prevent dominance of Pseudomonasdenitrificance (Kshirsagar, 1995). The final media compositionwas as follows: Nitrophenol (4-NP, 2,4-DNP and 2,4,6-TNP)200 mg/L, MgSO4 300 mg/L, CaCl2 62 mg/L, K2HPO4 800 mg/L, KH2
PO4 200 mg/L, FeCl3�6H2O 16 mg/L.The nitrophenol stock solution was prepared by dissolving 5 gm
of each nitrophenol in 100 mL acetone. The concentration of eachnitrophenol was adjusted in the feed depending upon the initialloading of nitrophenol intended during stepwise increase in accli-mation studies. For selecting aerobic denitrification mode over an-oxic denitrification (which is carried out in the conventional SBRsystem), changing of oxic–anoxic phases was prevented in the SBRsduring study. The feed provided pH around 7–7.5. Hybrid SBR sys-tem was proposed in the study, where both nitrification and deni-trification were achieved at reduced aeration (DO = 2 mg/L). TheDO was specifically maintained to achieve optimal nitrificationand denitrification by T. pantotropha. The selective feed playedimportant role in controlling autotrophic and heterotrophic nitri-fier and denitrifier populations in the bioreactors. Autotrophicnitrification (normal nitrifiers) requires inorganic nitrogen sourcesuch as NH4SO4 or KNO2. This was prevented in the present studywith selective feed which contained nitrophenol, as the sole sourceof nitrogen along with minimal media.
Elimination of inorganic nitrogen source gave selective pressureto promote heterotrophic nitrification and remove autotrophicnitrification in the beginning. In the study only partial nitrificationprocess was involved that included conversion of nitrite to nitrateafter breaking of nitrite from nitrophenol ring. Therefore role ofboth autotrophic and heterotrophic nitrifiers is limited to convertnitrite to nitrate and not ammonia to nitrate. Selective feed gaveselective advantage to predominate heterotrophic nitrifiers andaerobic denitrifiers. It facilitated initial breaking of nitrophenol tophenol and nitrite to proceed rapidly which in turn helped in rapidacclimation of the single sludge biomass to nitrophenols. However,the possibility of autotrophic nitrification was very limited in thepresent study and only partial autotrophic nitrification from nitriteto nitrate was possible. For it to proceed initial breaking of nitro-phenol ring should occur via heterotrophic nitrification with thesingle sludge containing T. pantotropha. However, within the het-erotrophic nitrifier populations to increase T. pantotropha popula-tion, MgSO4 concentration was increased from 100 to 300 mg/Lto selectively prevent dominance of P. denitrificance or other het-erotrophic nitrifiers (Kshirsagar, 1995).
P. Kulkarni / Bioresource Technology 128 (2013) 273–280 279
The single sludge containing T. pantotropha provided high ratesof nitrification and denitrification (SND) for the biodegradation ofnitrophenols. The presence and tentative proportion of T. pantotro-pha in the bioreactors was critical during present study. Duringpresent study, five key parameters were specifically adjusted to de-tect the presence and further quantitatively estimate T. pantotro-pha. These parameters can be used as direct evidence to identifyT. pantotropha from mixed microbial consortium.
3.3.1. The composition of biomassThe biomass composition was a critical factor for maintaining T.
pantotropha in high numbers in the bioreactors. The single sludgebiomass used during start up contained T. pantotropha biomassand sludge [Dairy sludge + cow dung (1:1)] in equal proportions(50% w/w of each). Several studies that have implemented bioaug-mentation strategies successfully, have used larger dosages of spe-cialized bacteria, around 25–32% w/w of the total biomass(Hernandez et al., 2012; Jianlong et al., 2002; Quan et al., 2004).The proportion of T. pantotropha used during the present studywas much higher compared to the other studies. This provides di-rect evidence of the presence and the dominance of T. pantotrophaover other microorganisms.
3.3.2. Extensive microscopic studiesExtensive microscopic studies were carried out to confirm the
presence and tentative percentage of T. pantotropha from the bio-reactors. In the phase contrast microscopy (Ziess optical imageanalysis microscope), T. pantotropha showed a non motile naturecompared to the highly motile nature of other microbes. FrequentGram staining was carried out to confirm the unique Gram nega-tive coccoidal morphology of T. pantotropha from the bioreactorsamples (Fig. 5).
3.3.3. DOThe nitrification and denitrification activities of T. pantotropha
are reported to be optimal at 25% of air saturation i.e. DO 2 mg/L(Robertson et al., 1988). Hence, DO was always kept 2 mg/Lthroughout the study in the bioreactors which specifically allowedthe growth of T. pantotropha over other nitrifying and denitrifyingbacteria.
3.3.4. Selective feedGupta and Kshirsagar (2000) reported the successful modifica-
tion of a plate count media to selectively enumerate T. pantotrophafrom the mixed culture and suppress the growth of Pseudomonasand other microorganisms. The modified medium contained highconcentration of MgSO4 from 100 to 300 mg/L. They also specifi-cally mentioned its use for quantitative estimation of T. pantotrophafrom the mixed microbial populations in the wastewater treatment.In the present study, the selective feed was specifically designed toselect T. pantotropha over the other nitrifiers and denitrifiers byadjusting the key nutrients e.g. MgSO4 from 100 to 300 mg/L. Thishelped in imposing the dominance of T. pantotropha over othermicroorganisms in the single sludge.
3.3.5. Biochemical studiesThe samples from the bioreactors were routinely screened using
plating method with general purpose media and selective media(High MgSO4 conc. 300 mg/L) for T. pantotropha to find out its ten-tative proportion (Gupta and Kshirsagar, 2000). Even though cul-turing and biochemical studies are traditional methods forisolation and identification of bacteria, they are still very accuratemethods and used extensively in microbial technology. Isolationand cultivation of filamentous bacteria in pure culture have beenfound to be promising methods to gain a better understanding ofbulking, foaming and biochemistry behind the filamentous bacte-
ria development (Bjornsson et al., 2002). Similarly, in the presentstudy, a detailed microbiological analysis and examination wascarried out to isolate and identify T. pantotropha from the bioreac-tor samples. The single sludge contained 60–70% population of T.pantotropha which was routinely confirmed with planting and bio-chemical tests specific to the species of T. pantotropha (Section 2.4).
The single sludge containing T. pantotropha, cow dung and dairysludge was able to sustain and degrade high concentrations ofnitrophenols (200 mg/L). Different members of food chains andfood webs such as bacteria, ciliates, flagellates, stalked flagellates,protozoa and rotifers could survive and proliferate in the bioreac-tors. This was detected by a Zeiss microscope with an image anal-ysis attachment under a phase contrast mode with 40�magnification. The presence of different members of food chainsand food webs shows the healthy state of single sludge in the pres-ence of toxic nitrophenols. In the beginning, addition of peptoneand yeast extract in the first month of start up, enhanced qualityof the flocculated single sludge biomass. The biomass was compactand showed good settling property during the acclimation of allthree nitrophenols reactors when compared with the biomass ofthe background control reactor (R). Microbiological examinationshowed good bacterial number when yeast extract and peptonewere added in the bioreactors. However populations of ciliatesand flagellates were equally predominant in the mixed singlesludge during acclimation. Rotifers were predominant during thelate phase of acclimation (nitrophenol concentration of 200 mg/Land a COD around 4500 mg/L).
In the present study, SND was accomplished with a specificallydesigned single sludge biomass containing T. pantotropha. Thisorganism offers great potential of SND which can take place withlow COD, low DO inputs with high conversion rates of ammoniato N2 (Robertson et al., 1988). The proportion of T. pantotrophawas specifically maintained in bioreactors (60–70% total bacterialpopulation) using selective feed. The plate count method and bio-chemical testing was routinely carried out to check T. pantotrophapopulation in the bioreactors. SND is a simple microbial processwith can be easily applied in any reactor system using basic micro-biological techniques which include various biochemical parame-ters like DO, pH, Temperature, selective media composition,addition or removal of key nutrient, etc. In microbial technology,when a particulate substrate (e.g. Nitrophenols) is degraded bymicroorganisms, it must be either applied as co-metabolite (co-metabolic mode) or sole substrate (direct substrate mode). In pres-ent study, SND was the sole mode of nitrogen removal in the SBR.Nitrophenols were used the as the sole source of nitrogen. Inor-ganic nitrogen sources (NH4SO4 or KNO2) were specifically re-moved from feed composition to prevent autotrophicnitrification. The operation of all four SBR was carried out atDO = 2 mg/L and anoxic phase was completely removed from SBRoperation to prevent autotrophic denitrification. Aerobic denitrifi-cation was the sole mode of denitrification as oxygen (DO) waspresent during denitrification process. SND was specifically ap-plied by using selective feed composition containing minimal med-ium with nitrophenol as the sole source of nitrogen and energywith DO = 2 mg/L throughout the study.
The SND (simultaneous heterotrophic nitrification and aerobicdenitrification) process described in the present manuscript is dis-tinctly different than the two step classical nitrification and deni-trification process. The classical process requires an aerobicenvironment for nitrification and an anoxic (anaerobic) environ-ment for denitrification. Therefore, the traditional or conventionalbiological process used in wastewater treatment to achieve nitro-gen removal involves separate aerobic and anaerobic phases usingseparate bioreactors or different aeration intervals. Some nitrifyingbacteria (e.g. T. pantotropha) are able to de-nitrify even under aer-obic conditions (Robertson and Kuenen, 1990). N2 is shown to be a
280 P. Kulkarni / Bioresource Technology 128 (2013) 273–280
volatile end product of these reactions i.e. under certain conditions(aerobic, but with limited oxygen supply), a direct conversion ofammonia is feasible. In this process (SND), ammonia is completelyde-hydrogenated via sequential oxidation to hydroxylamine andnitrite (nitrification) followed by sequential reduction to nitricoxide, nitrous oxide and dinitrogen (denitrification). Since the oxi-dation of ammonia to hydroxylamine requires molecular oxygen,the conversion to nitrogen by this route is obligatory aerobic anddepends on the ability of the organism to denitrify aerobically(Wehrfritz et al., 1993). Therefore, in the present study, SND inSBR using T. pantotropha was accomplished under aerobic condi-tion at DO = 2 mg/L. Robertson et al. (1988), showed that in T. pan-totropha, both nitrification and denitrification were increased asthe dissolved oxygen concentration fell until a critical level wasreached at approximately 25% of air saturation. At this point, therate of de-nitrification (aerobic) was equivalent to the anaerobicrate. Therefore, optimal SND using T. pantotropha can be achievedonly at 25% air saturation i.e. DO = 2 mg/L and not under anaerobicconditions.
4. Conclusions
The single sludge containing T. pantotropha was able to detoxifyand degrade nitrophenols under SND with optimal nitrophenol andnitrogen removal during acclimation studies. The specificallydeveloped single sludge biomass with T. pantotropha was responsi-ble for the optimal performance of SBR bioreactors. Nitrophenol asdirect source of nitrogen was found to be a promising approach forrapid acclimation and biomass development. Addition of simplesubstrate with vitamins and growth factors (yeast extract and pep-tone) improved the biomass growth. The selective feed helped indirecting SND in SBR.
Acknowledgements
I would sincerely like to thank Prof. S.R. Asolekar and Prof. S.K.Gupta for their kind support and help during present researchwork.
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