2,4-Dinitrotoluene removal in aerobic granular biomass sequencing batch reactors

International Biodeterioration & Biodegradation xxx (2016) 1e10

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2,4-Dinitrotoluene removal in aerobic granular biomass sequencing batch reactors G. Kiran Kumar Reddy a, b, M. Sarvajith a, Y.V. Nancharaiah a, b, *, V.P. Venugopalan a, b a b

Biofouling and Biofilm Processes Section, Water and Steam Chemistry Division, Bhabha Atomic Research Centre, Kalpakkam 603102, India Homi Bhabha National Institute, Anushakti Nagar Complex, Mumbai 400 094, India

a r t i c l e i n f o

a b s t r a c t

Article history: Received 30 July 2016 Received in revised form 7 October 2016 Accepted 25 October 2016 Available online xxx

Aerobic granules were cultivated in sequencing batch reactor (SBR) by feeding 2,4-dinitrotoluene (2,4DNT) along with acetate. Aerobic granules with an SVI10 of 34.57 ± 2.6 mL g
1 and average diameter of 0.78 ± 0.3 mm were formed during 30 d of SBR start-up period. In an alternative approach, aerobic granules cultivated using acetate as carbon source were acclimatized and evaluated for 2,4-DNT removal. In both the approaches, the aerobic granules exhibited rapid 2,4-DNT removal wherein >90% of 10 mg L
1 2,4-DNT was removed within 24 h cycle period. The aerobic granules also exhibited ammonium-nitrogen and phosphorus removal in addition to organic carbon removal, indicating that presence of 2,4-DNT did not negatively affect nutrient removal. In aerobic granular biomass reactors, most of the organic carbon was consumed within the first 6 h while, majority of the 2,4-DNT was removed during the 24 h cycle period. HPLC analysis detected smaller amounts of 2-amino-4nitrotoluene, a biotransformation product of 2,4-DNT. 2,4-DNT removal by granules under anaerobic conditions was observed to be much smaller compared to the aerobic SBR. Thus, 2,4-DNT removal by aerobic granules was likely mediated by combination of both oxidative and reductive pathways. Although, the mechanisms of 2,4-DNT removal requires further investigations, effective and stable removal of 2,4-DNT in aerobic granular biomass reactors offers practical possibilities for treatment of wastewaters from ammunition factories. © 2016 Elsevier Ltd. All rights reserved.

Keywords: Aerobic granular sludge Ammunition wastewater Dinitrotoluene biodegradation Explosives Nitroaromatic compounds

1. Introduction Activated sludge process (ASP) is the most widely employed aerobic wastewater treatment wherein microbial community grows in the form of suspended ‘flocs’ (or bioflocs) (van Loosdrecht and Brdjanovic, 2014). Besides aeration tank, ASP requires a settling tank for separation of flocculent sludge from the treated waters. Morgenroth et al. (1997) reported formation of aerobic granular biomass when column-type reactors were inoculated with activated sludge and operated in sequencing batch reactor (SBR) mode with bubbled-aeration and short settling times. Since then, aerobic granular biomass has attracted increased attention because of its great potential in transforming the future of municipal and industrial wastewater treatment plants (Sarma et al., 2016). The

* Corresponding author. Biofouling and Biofilm Processes Section, Water and Steam Chemistry Division, Bhabha Atomic Research Centre, Kalpakkam 603102, India. E-mail addresses: [email protected], [email protected] (Y.V. Nancharaiah).

dense microbial granules formed in these reactors can be rapidly separated from the treated water in the same reactor tank by gravity settling. Thus, the need for a separate settling tank becomes almost obsolete, therefore this new technology significantly minimizes plant footprint. In addition, aerobic granular biomass systems allow retention of high amount of biosolids in the reactor. Moreover, the granular biofilm structure of aerobic granules maintains different redox conditions i.e. aerobic, anoxic and anaerobic microenvironment in the granules which allows desired biological processes like organic carbon removal, ammonium oxidation, denitrification, and phosphorus removal to take place in wastewater treatment (de Kreuk et al., 2005). Although the mechanisms of formation of aerobic granules are not yet fully understood, environmental biotechnological applications of this novel microbial community are constantly evolving (Zhang et al., 2016; Sarma et al., 2016). Biodegradation of numerous xenobiotic compounds such as phenol (Tay et al., 2005a), p-nitrophenol (Suja et al., 2012), chlorinated phenols (Khan et al., 2011), pyridine (Adav et al., 2007), phthalic acids and esters (Zeng et al., 2008), tert-butyl alcohol (Tay

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et al., 2005b), chloroanilines (Zhu et al., 2011), metal chelating agents (Nancharaiah et al., 2006b), dyes (Kolekar et al., 2012), and organophosphorous esters (Kiran Kumar Reddy et al., 2014; Nancharaiah et al., 2015) by aerobic granular biomass has been demonstrated in laboratory scale reactors. Generally, xenobiotic compounds which support microbial growth have been used as the sole carbon source for cultivating aerobic granules (Adav et al., 2007; Tay et al., 2005b; Zeng et al., 2008). In the case of other xenobiotic compounds that did not support microbial growth, cultivation of aerobic granules can be achieved if they are supplied along with glucose or acetate (Khan et al., 2011; Kiran Kumar Reddy et al., 2014; Kolekar et al., 2012; Nancharaiah et al., 2006b, 2015; Suja et al., 2012; Zhu et al., 2011). Often, aerobic granules precultivated by feeding acetate are adapted for establishment of xenobiotic biodegradation (Carucci et al., 2008; Nancharaiah et al., 2008; Tay et al., 2005a). Removal of metal ions (Nancharaiah et al., 2010) and radionuclides (Nancharaiah et al., 2006a) has been demonstrated using native or chemically modified aerobic granules (Wang et al., 2015; Suja et al., 2014). Denitrification of nitrate was also significant in aerobic granular biomass reactors (Nancharaiah and Venugopalan, 2011; Suja et al., 2015). Reductive precipitation of soluble and toxic Cr(VI) to less soluble Cr(III) was demonstrated for remediation of chromate contaminated waters (Nancharaiah et al., 2010). Dynamic community and diverse metabolic capabilities of microbial granules allows the treatment of various xenobiotic compounds. Occurrence of nitroaromatic compounds in the atmosphere, terrestrial and aquatic environments is attributed to agricultural, military and industrial activities (Boopathy et al., 1998). Nitroaromatic compounds are widely used in chemical synthesis, manufacturing of explosives, herbicides, fungicides, insecticides, polyurethane foam and dyes (Boopathy et al., 1994; Hughes et al.,
et al., 2015; Vanderloop et al., 1999). Nitro1999; Podlipna toluenes (2- and 4-nitrotoluenes) and dinitrotoluenes (2,4dinitrotoluene (2,4-DNT); 2,6-dinitrotoluene (2,6-DNT)) are the by-products in manufacturing explosives i.e. 2,4,6-trinitrotoluene (TNT) (Hughes et al., 1999; Anand and Celin, 2017). Due to toxicity, carcinogenicity and persistence in the environment, 2,4DNT, 2,6-DNT and TNT are categorized as priority pollutants by the US EPA. Thus, technologies for effective removal of these compounds from effluents originating from manufacturing plants and polluted sites is required to avoid release, transport and toxicity to biota in the environment (Clark and Boopathy, 2007; Anand and Celin, 2017). Use of ineffective technologies and improper disposal practices has led to the release of these nitroaromatics and contamination of soil and groundwater near ammunition manufacturing facilities (Boopathy, 2000). Adsorption by activated carbon and incineration of exhausted carbon is currently practised for treating 2,4-DNT contaminated wastewater (Vanderloop et al., 1999). However, this method is expensive and also causes air pollution (Snellinx et al., 2002). Microbial technologies are promising for remediation because of the effective biotransformation and biodegradation capabilities. Several bacterial strains such as Arthrobacter sp. (Küce et al., 2015), Rhodococcus pyridinivorans NT2 (Kundu et al., 2015), Burkholderia sp. (Nishino et al., 2000), and Pseudomonas sp. (Spanggord et al., 1991) have been reported to be capable of 2,4-DNT biodegradation. 2,4-DNT removal by microorganisms was observed under aerobic, anoxic and anaerobic conditions (Huang et al., 2015; Kundu et al., 2015; Noguera and Freedman, 1996; Vanderloop et al., 1999). Coupled aerobic and anaerobic systems have been also studied for effective 2,4eDNT removal (Wang et al., 2011). Removal mechanisms of 2,4-DNT by microorganisms involves biodegradation (Kundu et al., 2015), biotransformation (Huang et al., 2015) or both (Freedman et al., 1996; Wang et al., 2011).

Previous studies on 2,4-DNT removal were carried out using axenic bacterial cultures, activated sludge and biofilms (Wang et al., 2011). Though aerobic granules have been reported to be superior to activated sludge for biodegradation of toxic and recalcitrant compounds (Zhang et al., 2016), no studies pertaining to use of aerobic granules in treating wastewater containing explosives have been reported. In this study, the effectiveness of aerobic granular biomass SBRs for removal of 2,4-DNT was investigated for the first time. Experiments were carried out in one litre volume bubble column SBRs containing aerobic granules cultivated under 2,4-DNT enrichment conditions in the presence of acetate or lactate. In addition, 2,4-DNT removal was studied in serum bottles to determine the biotransformation potential of aerobic granules under anaerobic conditions. 2. Materials and methods 2.1. Cultivation of aerobic microbial granules A bubble-column glass reactor with a total volume of 1.6 L (total height: 42 cm, diameter: 6.5 cm) was used with 1 L working volume (working height: 30 cm, diameter: 6.5 cm) for the cultivation of microbial granules (Fig. 1A). The effective height to diameter (H/D) ratio of the reactor was 4.6. Activated sludge collected from the aeration tank of an operating domestic wastewater treatment plant located at Kalpakkam, India was used as the seed sludge. Reactor was inoculated with 0.25 L activated sludge and operated in SBR mode. The SBR (hereafter, SBR-I) was fed with simulated wastewater (SWW) prepared in deionised water containing the following constituents (in mM): sodium acetate (6.3), MgSO4$7H2O (0.36), KCl (0.47), NH4Cl (3.54), K2HPO4 (0.42), KH2PO4 (0.21), and CaCl2$2H2O (0.25). 1 mL of trace elements mix (Nancharaiah et al., 2008) was added to 1 L SWW. 2,4-DNT was added from a stock solution (200 mg L
1 2,4-DNT) to obtain a final concentration in the range of 5e10 mg L
1 2,4-DNT in the SBR (Table 1). The SBR was operated in 24 h cycle period which consisted of 10 min fill, 23 h aeration, 5 min settle, 10 min decant, and 45 min idle periods. During aeration period, the reactor was supplied with compressed air at the bottom of SBR through a porous stone at a superficial air velocity of 1.2 cm s
1. The SBR was operated with 70% volumetric exchange ratio in a temperature (~30  C) controlled room. A port located at 9 cm from the bottom was used for decanting the treated water at the end of the cycle period. 2.2. Operating performance of SBR fed with acetate and 2,4-DNT After formation of microbial granules, reactor performance was monitored. The SBR-I was fed with SWW containing acetate and 2,4-DNT (10 mg L
1). Samples were collected at the beginning and end of the cycle periods for monitoring reactor performance. Occasionally, samples were also collected at regular time intervals during the cycle period. Samples were centrifuged at 8000 rpm for 10 min to remove the suspended cells and stored at 4  C until further analysis. The samples were appropriately diluted and ana

lysed for total organic carbon (TOC), 2,4-DNT, NHþ 4 -N, NO3 -N, NO2 N and PO3
-P. Samples were also analysed for putative 2,4-DNT 3 intermediates such as aminonitrotoluene isomers and diaminotoluenes. The substrate utilisation pattern was indirectly monitored online using a dissolved oxygen (DO) probe (Hach, USA). 2.3. Removal of 2,4-DNT by aerobic microbial granules cultivated by feeding only acetate Aerobic microbial granules were harvested from an operating laboratory scale SBR treating acetate-containing SWW (without

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Fig. 1. Schematic representation of SBR used for studies on 2,4-DNT removal by aerobic microbial granules (A) Aerobic granules cultivated by feeding acetate plus 2,4-DNT (B) Aerobic granules cultivated by feeding acetate as the sole carbon source (C). Scale bar in (B) and (C) ¼ 5 mm.

Table 1 Operational strategy of SBRs used for development of 2,4-DNT removing aerobic granules. Parameter

SBR-I

SBR-II

Chief carbon source TOC contribution 2,4-DNT Seed biomass in the SBR

Acetate 150 mg L
1 as C 5e10 mg L
1 Activated sludge

Lactate 150 mg L
1 as C 10 mg L
1 Acetate fed aerobic granules

2,4-DNT) (Nancharaiah and Venugopalan, 2011) and evaluated for 2,4-DNT removal. In brief, these granules were cultivated in a 3 L SBR operating with 6 h SBR cycle period with 4 cycles per day. Each cycle had 4.5 h aeration period and the composition of SWW used was the same as mentioned above for SBR-I, except 2,4-DNT. The granular biomass cultivated using acetate-containing SWW was collected and exposed to 10 mg L
1 of 2,4-DNT for acclimatization (Table 1). The biomass was directly added into shake flasks and incubated in an orbital shaker at 30  C and 100 rpm. The acclimation was continued for 10 days during which the feed was replaced at the end of every 24 h period with SWW containing lactate (4.1 mM as sodium lactate) and 10 mg L
1 2,4-DNT. At the end of 10 d, the entire biomass was transferred into another 1 L bubblecolumn SBR (SBR-II) and operated in SBR mode. The SBR-II was fed with SWW containing lactate and 10 mg L
1 2,4-DNT and operated with 24 h cycle period and 70% volume exchange ratio. Samples were collected at the beginning and end of the SBR cycle period and also during the cycle period to monitor reactor performance in terms of TOC and 2,4-DNT removal. DO profiles were recorded online during the SBR cycle period.

2.4. Removal of 2,4-DNT by granular biomass in anaerobic conditions Experiments were setup in serum bottles to evaluate 2,4-DNT removal under anaerobic conditions. Granular biomass was harvested from SBRs fed with acetate plus 2,4-DNT (SBR-I) and acetate alone. Biomass (2 g wet weight corresponding to 5.1 g MLSS L
1) was directly added to each of the 130 mL serum bottle containing 80 mL of SWW with acetate or lactate plus 10 mg L
1 2,4-DNT. Serum bottles were sparged with argon gas for 5 min, crimp-sealed with butyl rubber septa and incubated in an orbital shaker at 30  C and 100 rpm. After 24 h, the medium was removed completely and replaced with the respective SWW containing acetate or lactate plus 10 mg L
1 2,4-DNT. This process was repeated for 5 cycles and samples were collected during each cycle period at every 12 h. The samples were analysed for 2,4-DNT and possible transformation intermediates.

2.5. HPLC Dionex UltiMate 3000 HPLC e variable wavelength detector system (ThermoScientific, USA) was used for quantifying 2,4-DNT and intermediates. ODS Hypersil C18 (5 mm) column was used for the separation of 2,4-DNT and other intermediates. For the detection and quantification of 2,4-DNT, 2-amino-4-nitrotoluene (2-A-4NT) and 4-amino-2-nitrotoluene (4-A-2-NT), 50% of HPLC grade methanol was used as the eluent (Oh and Chiu, 2009). The eluent flow rate was 0.8 mL min
1 and the detection was at 254 nm. 2,4diaminotoluene (2,4-DAT) was estimated using the same column with acetonitrile: phosphate buffer (20 mM, pH 7) in 30:70 ratio at a flow rate of 1 mL min
1. 2,4-DAT was detected at 224 nm (Oh and

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Chiu, 2009). 2.6. Analytical methods High temperature combustion followed by infrared based detection of the liberated CO2 was used for TOC estimation. TOC was measured using TOC-VCSH analyser (Shimadzu, Japan) equipped with an autosampler. Samples after appropriate dilution were injected into the combustion chamber. The analyser operates at 600  C and high purity nitrogen (99.995%) was used as the carrier gas. Luminescent dissolved oxygen (LDO) sensor probe (Hach, USA) connected to a portable multi-parameter device (Hach, USA) was used for online DO monitoring. Nitrate was estimated by HPLC method and nitrite was estimated by N-(1-naphthyl)ethylenediamine dihydrochloride method using a UVeVis spectrophotometer (Nancharaiah and Venugopalan, 2011). Inorganic phosphorus was measured by standard ascorbic acid method and ammonia was measured by phenate method (APHA, 2005). Biomass characteristics such as average diameter, mixed liquor suspended solids (MLSS), volatile suspended solids (VSS), and sludge volume index (SVI) were measured as per the standard methods (APHA, 2005). Granules were homogenised and extracted for extracellular polymeric substances (EPS) using alkaline heating method (1 M NaOH, pH 11 and 80  C for 2 h) (McSwain et al., 2005). Total carbohydrates and total proteins in the extracted EPS were estimated by standard phenol-sulphuric acid reagent and biuret reagent, respectively.

carbon source, did not support granulation, while the use of pnitrophenol along with acetate enabled successful cultivation of aerobic granules capable of p-nitrophenol biodegradation. Nevertheless, presence of xenobiotic compounds during the granulation process can serve as a selection pressure to enrich the microbial population capable of tolerating and biodegrading the xenobiotic. This approach has been used for the cultivation of aerobic microbial granules for the degradation of persistent organics such as tributyl phosphate (Nancharaiah et al., 2015), nitrilotriacetic acid (Nancharaiah et al., 2006b), and 2-chlorophenol (Khan et al., 2011). Xenobiotics such as nitroaromatic compounds are persistent, not

3. Results and discussion 3.1. Granule cultivation and performance of SBR-I fed with acetate and 2,4-DNT The activated sludge used for the inoculation of SBR was dominated by filamentous microorganisms. During the course of bubble-column SBR operation, the activated sludge gets transformed initially to minute aggregates and then to compact and dense millimetre sized granules. Often the time required for granulation depends on various factors such as the type of carbon source, organic loading rate, type of xenobiotics present in the reactor and other factors like reactor configuration and superficial air velocity. Granulation occurs within a few days if the reactor is operated under optimum operational conditions with multiple cycles in a day by feeding labile carbon sources (e.g., acetate) which provides carbon, energy and electrons required for the growth of microorganisms and development of multispecies microbial aggregates. When the provided organic carbon is not easily metabolized, the granulation may take longer time due to poor microbial growth. Granulation cannot be realised when compounds which does not support the microbial growth are used as the sole carbon source. In such cases, the target xenobiotic compound is supplied along with a labile carbon source such as acetate and glucose which supports the microbial growth and granulation. For example, Suja et al. (2012) showed that para-nitrophenol, when used as the sole

Table 2 Characteristics of 2,4-DNT removing aerobic granules after 30 days of reactor operation. Parameter

SBR-I

SBR-II

Diameter (mm) Mixed Liquor Suspended Solids (MLSS) (g L
1) Volatile Suspended Solids (VSS) (g L
1) Sludge Volume Index (SVI10) (mL g
1) Total carbohydrates (mg g
1 wet weight) Total proteins (mg g
1 wet weight)

0.78 (±0.32) 1.88 (±0.49) 1.01 (±0.22) 34.57 (±2.60) 0.76 (±0.09) 98.20 (±6.18)

2.85 (±1.14) 0.75 (±0.26) 0.56 (±0.161) 53.30 (±3.10) 1.65 (±0.15) 39.28 (±4.69)

Fig. 2. Removal performance of SBR-I fed with acetate and 10 mg L
1 2,4-DNT. (A) Removal of 2,4-DNT and total organic carbon (TOC) in SBR from 31 to 45 d of operation. (B) 2,4-DNT and TOC removal profiles in two representative SBR cycles. (C) Dissolved oxygen (DO) profiles in two representative SBR cycles.

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easily biodegraded and poorly support microbial growth. Therefore, the use of labile substrates such as acetate or lactate is justified. 2,4-DNT is resistant to aerobic biodegradation due to the presence of strong electron withdrawing nitro groups. Thus, acetate was used along with 2,4-DNT for cultivation of 2,4-DNT degrading aerobic granules. The wastewater generated from ammunition factories generally contain organics such as ethanol, ether, acetone or their degradation productions at much higher concentrations than 2,4-DNT (Christopher et al., 2000). These organic carbon sources can support microbial growth and granulation when real ammunition wastewater is treated using SBRs. Bioreactor systems using axenic bacterial cultures and biofilm systems have been evaluated for the treatment of explosives contaminated wastewater (Christopher et al., 2000; Wang et al., 2011). In the case of remediation of soils contaminated with explosive compounds, both in situ and ex situ bioremediation methods are considered. Use of bacteria (Gumuscu and Tekinay, 2013), fungal strains (Anasonye et al., 2015) and the soil slurry reactor (Boopathy, 2000) for the remediation of contaminated sites has been reported. The activated sludge flocs used for inoculation of SBR-I were in black colour and had an average size of 60 mm. Formation of compact and tiny granules was noticed during SBR operation with simulated wastewater containing acetate and 2,4-DNT. The black colour of the seed sludge disappeared and the granules became in light orange in colour. The size of the granules increased and reached a steady-state within one month of SBR start-up period. The morphology of the granules on day 30 was shown in Fig. 1. The granules harvested after 30 d of SBR-I operation had an average diameter of 0.78 (±0.32) mm (Fig. 1B). The physico-chemical characteristics of the aerobic granules are shown in Table 2. The SVI10 of the seed sludge was 330 mL g
1 indicating poor settleability. However, the SVI10 of the granules formed in the SBR was 34.57 ± 2.6 mL g
1, indicating excellent settling properties. The reactor was initially fed with acetate plus 5 mg L
1 2,4-DNT. After two weeks of start-up period, formation of tiny granules was visible, after which the concentration of 2,4-DNT was increased to 10 mg L
1 keeping acetate concentration constant. After achieving steady-state granulation in SBR-I within 30 days, removal of TOC and 2,4-DNT was monitored over a period of 15 SBR cycles (Fig. 2A). It is clearly evident that most of the added organic carbon was removed by the end of 24 h cycle period. TOC removal was consistently >90%. Interestingly, 2,4-DNT removal was also more than 90%. TOC and 2,4-DNT removal profiles in independent 24 h SBR cycle periods showed that most of the TOC was removed within the first 3 h of cycle period (Fig. 2B). Acetate being the major source of TOC which is easily metabolizable under bubble-aeration conditions, the changes in the dissolved oxygen profiles (Fig. 2C) could be corroborated to the TOC removal pattern. After majority of the TOC was removed, the DO in the bulk liquid reached the saturation phase. In contrast, 2,4-DNT removal was noticed throughout the SBR cycle period (Fig. 2B). In control experiments performed under similar experimental conditions, but without the biomass showed negligible 2,4-DNT removal (data not shown). These results for the first time demonstrate cultivation of 2,4-DNT removing aerobic granules in SBR by feeding acetate and 2,4-DNT. The present study extends the environmental biotechnological applications of aerobic granular biomass technology for the treatment of 2,4-DNT containing wastewaters (e.g., ammunition wastewater).

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glucose) have been acclimatized for developing xenobiotic degradation. This approach is aimed at decreasing the start-up time required for the cultivation of aerobic granules (Tay et al., 2005a). Presence of a wide diversity of microorganisms, and the inherent dynamic nature of aerobic granules allows establishment of catabolic pathways for the degradation of xenobiotics (Zhang et al., 2016). This approach has been already described for rapid cultivation of aerobic granules capable of biodegradation of phenol (Tay et al., 2005a), 4-chlorophenol, and 2,4,6-trichlorophenol (Carucci et al., 2008). Acetate fed aerobic granules harvested from an operating 3 L volume SBR were evaluated for 2,4-DNT removal. The aerobic granules cultivated using acetate were acclimated to lactate plus

3.2. 2,4-DNT removal by pre-cultivated aerobic microbial granules In an alternative approach, multi species aerobic microbial granules cultivated using labile carbon sources (i.e. acetate and

Fig. 3. Performance of SBR-II fed with lactate and 2,4-DNT. (A) Total organic carbon and 2,4-DNT removal during 15 d of SBR operation. (B) TOC, 2,4-DNT removal profiles during two representative SBR cycles. (C) DO profiles in two representative SBR cycles.

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2,4-DNT for 10 days. Lactate was used in place of acetate in order to evaluate the role of other labile carbon source, if any, on 2,4-DNT removal performance. After the acclimation period, granules were transferred to SBR-II. Morphology and physicochemical characteristics of the aerobic granules from SBR-II (Table 2) are presented in Fig. 1C and Table 2. Removal of TOC and 2,4-DNT in SBR-II is presented in Fig. 3A. Interestingly, removal performance of SBR-II inoculated with pre-cultivated granules was similar to that of SBR-I (acetate þ 2,4-DNT) and more than 90% of the supplied TOC and 2,4-DNT were removed in 24 h cycle period. TOC and 2,4-DNT removal profiles in two independent cycles are shown in Fig. 3B. Majority of the 2,4-DNT was removed within the first six hours when the lactate was still available in the medium. However, further experimentation is needed to confirm if 2,4-DNT removal involves any co-metabolism. TOC removal profiles were in

agreement with the DO profiles (Fig. 3C) as within 6 h, most of the TOC was removed, which was followed by DO saturation. These studies indicate the inherent metabolic diversity of aerobic microbial granules to utilise different labile carbon sources such as acetate or lactate and also to quickly acclimatise themselves for the removal of xenobiotics such as 2,4-DNT. The changes in the microbial community responsible for the acclimatization to remove 2,4-DNT requires community analysis before and after the 2,4-DNT exposure. The studies related to community profiling and isolating bacterial strains responsible for 2,4-DNT removal are currently being pursued. 3.3. Nutrient removal in SBRs in the presence of 2,4-DNT In addition to TOC removal, removal of nutrients such as

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Fig. 4. Nutrient removal in SBRs. Removal of NHþ 4 -N and PO3 -P in SBR-I (A) and SBR-II (B). In SBR-II, cycles 1e5 at the beginning are without 2,4-DNT and 5e10 cycles are in the presence of 10 mg L
1 2,4-DNT.

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nitrogen and phosphorus is an essential requirement of wastewater treatment. Therefore, removal of ammonium-nitrogen and phosphorus was monitored in both the SBRs receiving acetate plus 2,4DNT (SBR-I) or lactate plus 2,4-DNT (SBR-II). Nearly 50% of the added ammonium-nitrogen was removed in both the SBRs (Fig. 4). Phosphorus removal was superior in SBR-I as compared to SBR-II. The removal of ammonium-nitrogen and phosphorus in control reactors fed either with acetate or lactate (without 2,4-DNT) was similar to the nutrient removal performance in SBR I and SBR II (Fig. 4B). In fact, the removal of inorganic phosphate was higher in SBR-I fed with acetate plus 2,4-DNT as compared to SBR fed only with either acetate or lactate (control). The data on nutrient removal strongly suggested that presence of 2,4-DNT in wastewater does not have any negative effect on nutrient removal in aerobic granular biomass SBRs. 3.4. 2,4-DNT removal in anaerobic serum bottle experiments Anaerobic transformation of 2,4-DNT by different bacterial strains and mixed cultures was reported (Huang et al., 2015; Hughes et al., 1999; Wang et al., 2011). Recently, growth of an obligate marine strain, Shewanella marisflavi EP1 under anaerobic conditions coupled to the reduction of 2,4-DNT through dissimilatory reduction was reported (Huang et al., 2015). The metal reducing Shewanella was able to thrive by using lactate and 2,4DNT as electron donor and acceptor, respectively. Authors have reported complete reduction of 18.2 mg L
1 of 2,4-DNT within 24 h by the growing cells of S. marisflavi EP1. In an another study, mixed culture anaerobic filter reduced most of the supplied 2,4-DNT using ethanol as the electron donor (Wang et al., 2011). In both the studies, the reduction products detected were 2-amino-4nitrotoluene (2-A-4-NT), 4-amino-2-nitrotoluene (4-A-2-NT), and 2,4-diaminotoluene (2,4-DAT). The aerobic granules from SBR-I and acetate fed SBR were incubated with acetate or lactate respectively, each with 10 mg L
1 2,4-DNT in anaerobic conditions. The removal of 2,4-DNT over a period of 5 cycles is shown in Fig. 5. The biomass which was regularly exposed to 2,4-DNT in SBR-I was also poor in 2,4-DNT removal in anaerobic conditions (Fig. 5A). Although the biomass content in the serum bottles was high (5 g MLSS L
1) compared to the biomass content in the SBRs (~2 g MLSS L
1), 2,4-DNT removal was poor in anaerobic conditions. Even after 24 h incubation, almost 60% of the supplied 2,4-DNT remained unused in the medium. Similar observations were made when the acetate fed precultivated granules were incubated with 2,4-DNT under anaerobic conditions. When estimated for reduction intermediates, only small quantities of 2-A-4-NT were detected and the concentrations were below 1 mg L
1. The results suggest that 2,4-DNT removal is only marginal under anaerobic conditions. Thus, the mechanisms accounting for removing majority of the 2,4-DNT in aerobic granular biomass SBRs do not appear to follow reductive pathway.

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carbon source (ethanol) for aerobic 2,4-DNT degradation, nearly 41% decrease in nitrite release was observed. Therefore, presence of high concentrations of these labile carbon sources led to the reduction of 2,4-DNT to form aminotoluenes (Freedman et al., 1996). In oxidative degradation pathway, ring cleavage can be initiated by the oxygenases or the peroxidase enzymes to eventually produce tricarboxylic acid cycle intermediates, finally leading to formation of nitrite, CO2 and biomass (Kundu et al., 2015). The released nitrite can also serve as the nitrogen source for certain microorganisms (Monti et al., 2005). The aerobic degradation of 2,4-DNT by Pseudomonas sp. is initiated by a dioxygenase enzyme attack to replace nitro group with hydroxyl group (Spanggord et al., 1991). This leads to the release of nitrite and formation of 4-methyl5-nitrocatechol (4-M-5-NC). Conversion of 4-M-5-NC to tricarboxylic acid cycle intermediates is mediated by a set of oxygenases and hydrolases as reported in Burkholderia cepacia (Monti et al., 2005). Simultaneously, 2,4-DNT is subjected to sequential reduction reactions via reductive pathway, wherein nitro groups of 2,4DNT are reduced to form aminonitrotoluene isomers, and finally to diaminotoluenes (2,4-DAT). The aminonitrotoluenes or diaminotoluenes are subjected to further degradation. However, the exact pathways are yet to be identified. HPLC was used for the detection and identification of the intermediates namely, 2-A-4-NT, 4-A-2-NT and 2,4-DAT. Representative chromatograms obtained for samples collected during SBR cycle period at 0 h, 6 h and 24 h are shown in Fig. 6. Sample

3.5. Intermediates formed during 2,4-DNT degradation and the possible mechanism Biological removal of 2,4-DNT was studied by estimating the intermediates formed during the biodegradation or transformation. In anaerobic conditions, 2,4-DNT removal is mainly attributed to the biotransformation to produce aminonitrotoluene isomers (2-A-4NT, 4-A-2NT) and diaminotoluenes (2,4-DAT) (Huang et al., 2015). In aerobic conditions, 2,4-DNT removal occurs by combination of oxidative or reductive pathways. In a study, active biomass collected from munition wastewater treatment plant was able to degrade 2,4-DNT with the stoichiometric release of nitrite. When the same biomass was tested along with labile

Fig. 5. 2,4-DNT removal by granular biomass in serum bottles under anaerobic conditions. Anaerobic removal of 2,4-DNT by aerobic granules harvested from SBR-I and fed with acetate and 2,4-DNT (A). Anaerobic removal by the pre-cultivated aerobic granules fed with lactate and 2,4-DNT (B).

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collected immediately (0 h) after feed addition to the SBR had only 2,4-DNT peak (retention time (Rt) 10.5 min). A transient peak was observed at Rt 12.7 min in the 6 h sample. In the 24 h sample, both 2,4-DNT peak and the transient peak were disappeared. A stable 2A-4-NT peak (Rt 7 min) was appeared in 24 h samples. The concentration of 2-A-4-NT formed in representative cycles from SBR-I and SBR-II are shown in Fig. 7. 4-A-2-NT and 2,4-DAT were not detected during the cycle period and also in the effluent samples of SBR-I and SBR-II. Although 2A-4-NT was formed, its concentration was not in stoichiometric relation to the amount of 2,4-DNT removed in the SBRs. Maximum concentrations of 2A-4-NT detected were ~1 mg L
1 which is <10% of the total 2,4-DNT supplied. Around 1 mg L
1 of the supplied 2,4-DNT was transformed into a potentially toxic intermediate, 2-amino-4-nitrotoluene. Various toxicity assays using the test organisms have shown that the reduced intermediates are more toxic than the parent compound (Dodard et al., 1999). In a study with test bacterium Vibrio fischeri, the IC50 value for 2-A-4-NT was reported to be 14.28 mg L
1 (94 mM), whereas for 2,4-DNT it was 48.95 mg L
1 (269 mM), indicating the greater toxicity of 2-A-4-NT over 2,4-DNT. In the current study, the concentration of residual 2-A-4-NT was at 1 ± 0.2 mg L
1, much below the IC50. However, there is a need to further optimize the treatment system to completely eliminate 2A-4-NT from the treated waters. Reduction reactions are possible in bubble-column aerobic granular biomass reactors due to the presence of different redox microenvironments i.e. aerobic, anoxic and anaerobic zones within the granules. Denitrification is commonly achieved in aerobic granular biomass reactors, thus, nonspecific action of nitroreductases of abundant denitrifying microorganisms (Nancharaiah and Venugopalan, 2011; Adav et al., 2010) can account for the partial reduction of 2,4-DNT. 2-A-4-NT was formed only in minor amounts. The other possibility for the removal of the rest of the 2,4DNT is by oxidative degradation pathway involving catechol intermediates. The effluents from 2,4-DNT receiving SBRs were completely devoid of nitrate; but small quantities of nitrite was

present. Nitrite was absent in the effluent samples of SBR operated without 2,4-DNT. But, as the granules represent mixed consortium of microorganisms including nitrifying bacteria, nitrite formation from ammonia oxidation is possible. Additional experiments using 2,4-DNT as the sole nitrogen source can explain the origin of nitrite release. Based on the obtained results from 2,4-DNT removal by aerobic granules under aerobic and anaerobic conditions, preliminary data on intermediates and the previously reported biodegradation mechanisms, a 2,4-DNT biodegradation pathway by aerobic microbial granules is proposed (Fig. 8). Majority of the 2,4DNT is removed by aerobic oxidation, while smaller quantities of 2A-4-NT is formed by biotransformation. Further investigations are being continued to decipher microbial community of 2,4-DNT removing aerobic granules, identify degradative organisms and to unravel 2,4-DNT removal mechanisms.

3.6. Practical implications 2,4-DNT is a priority pollutant and common contaminant of soil, water at ammunition manufacturing and storage sites. Microorganisms can employ oxidative and reductive pathways for biodegradation and biotransformation, respectively for removing 2,4-DNT. Often, combination of both aerobic and anaerobic conditions facilitate effective biological removal of nitroaromatic compounds. In this study, we demonstrated that aerobic granules can be cultivated in the presence of 2,4-DNT and utilized for effective removal of 2,4-DNT in sequencing batch reactors. Since 2,4-DNT is recalcitrant and poorly supports microbial growth, supply of other easily metabolizable carbon sources is necessary for cultivation of aerobic granules. Presence of co-existing aerobic, anoxic and anaerobic microorganisms within microenvironments of aerobic granules even during aeration period facilitates both biodegradation and biotransformation of dinitrotoluenes. Moreover, sequencing batch reactors allows incorporation of alternating aerobic-anaerobic phases in the cycle period. Thus, aerobic granular biomass sequencing batch reactors offers a better choice for

Fig. 6. Overlaid chromatograms obtained for SBR samples at different time intervals. 1 refers to the spectra for 0 h sample, showing only the 2,4-DNT peak (peak A at Rt ¼ 10.5 min). 2 refers to spectra for 6 h sample showing a decrease in response for peak A and the appearance of two new peaks at 12.7 min (peak B) and 7 min (peak C belong to 2-amino-4nitrotoluene). 3 refers to spectra for effluent sample, showing disappearance of peak A (for 2,4-DNT) and peak B and a slight increase in the response for peak C (2-amino-4nitrotoluene).

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remediation of waters contaminated with nitroaromatic compounds. Future studies should focus on mechanisms of nitroaromatic compounds removal, removal of more recalcitrant trinitrotoluene and removal of these nitroaromatic compounds from real effluents. 2,4-DNT removal was rapid in the aerobic granular biomass SBRs. However, removal of 2,4-DNT was not complete and always associated with a residual 2,4-DNT in the treated water. It is unclear why there should be a residual of 2,4-DNT in the aerobic granular sludge SBR. Further studies aimed at 1) long term reactor operation for further enrichment 2) operation of reactor with higher hydraulic retention time and 3) integration of 2,4-DNT degrading strains into aerobic granules through bioaugmentation are necessary for improving 2,4-DNT removal efficiency. Integration of additional post-treatment step based on adsorption or reverse osmosis may be considered, if removal efficiency of the biological treatment cannot be improved to achieve the maximum non-toxic concentrations of 0.13 mg 2,4-DNT L
1. However, this will significantly add to the overall cost of the treatment process.

4. Conclusions Aerobic granular biomass capable of 2,4-DNT removal was successfully cultivated from activated sludge in a sequencing batch reactor by feeding 2,4-DNT along with acetate. In an alternative approach, pre-cultivated aerobic granules using acetate were acclimated for 2,4-DNT removal. Aerobic granules obtained from

Fig. 7. Formation and build-up of 2-amino-4-nitrotoluene during 10 mg L
1 2,4dinitrotoluene removal in representative cycles in SBRs. SBR-1 received 10 mg L
1 2,4-DNT along with acetate, while SBR-II received 10 mg L
1 2,4-DNT along with lactate.

Fig. 8. Proposed mechanisms of 2,4-dinitrotoluene removal by aerobic granules. It was based on measurements and biodegradation information taken from the available literature. D ¼ Detected. N.D. ¼ Not detected. N.S. ¼ Not studied. (2,4-DNT: 2,4dinitrotoluene, 2-A-4-NT: 2-amino-4-nitrotoluene, 4-A-2-NT: 4-amino-2nitrotoluene, 2,4-DAT: 2,4-diaminotoluene. 4-M-5-NC: 4-methyl-5-nitrocatechol).

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both the approaches were able to effectively remove 2,4-DNT when fed along with acetate or lactate. Removal of 2,4-DNT by aerobic granular biomass is most likely mediated through both oxidative and reductive pathways. In oxidative pathway, 2,4-DNT can be acted upon by the oxygenases and ultimately converted to carbon dioxide and water. Small quantities of 2-amino-4-nitrotoluene detected in the effluents can arise from reduction of 2-nitro group of 2,4-DNT by non-specific action of nitroreductases. This is the first study, on cultivation of aerobic granules capable of 2,4-DNT removal. Interestingly, removal of ammonium-nitrogen and phosphorus in the sequencing batch reactors was not negatively influenced by the presence of 2,4-DNT in sequencing batch reactors. The approach described here can be explored further for development of aerobic granular biomass technology for treating ammunition wastewaters. References Adav, S.S., Lee, D.J., Lai, J.Y., 2010. Microbial community of acetate utilizing denitrifiers in aerobic granules. Appl. Microbiol. Biotechnol. 85, 753e762. Adav, S.S., Lee, D.J., Ren, N.Q., 2007. Biodegradation of pyridine using aerobic granules in the presence of phenol. Water Res. 41, 2903e2910. €nen, M., Kontro, J., Bjo € rklo €f, K., Vasilyeva, G., Anasonye, F., Winquist, E., R€ asa Jørgensen, K.S., Steffen, K.T., Tuomela, M., 2015. Bioremediation of TNT contaminated soil with fungi under laboratory and pilot scale conditions. Int. Biodeterior. Biodegrad. 105, 7e12. APHA, 2005. Standard Methods for the Examination of Water and Wastewater, twenty-first ed. APHA, Washigton, DC. Anand, S., Celin, S.M., 2017. Green technologies for safe disposal of energetic materials in the environment. In: DeLuca, L.T., et al. (Eds.), Chemical Rocket Propulsion. Springer Aerospace Technology. http://dx.doi.org/10.1007/978-3-31927748-6_35. Boopathy, R., 2000. Bioremediation of explosives contaminated soil. Int. Biodeterior. Biodegrad. 46, 29e36. Boopathy, R., Wilson, M., Montemagno, C.D., Manning, J.F., Kulpa, C.F., 1994. Biological transformation of 2,4,6-trinitrotoluene (TNT) by soil bacteria isolated from TNT-contaminated soil. Bioresour. Technol. 47, 19e24. Boopathy, R., Kulpa, C.F., Manning, J., 1998. Anaerobic biodegradation of explosives and related compounds by sulfate-reducing and methanogenic bacteria: a review. Bioresour. Technol. 63, 81e89. Carucci, A., Milia, S., De Gioannis, G., Piredda, M., 2008. Acetate-fed aerobic granular sludge for the degradation of chlorinated phenols. Water Sci. Technol. 58, 309e315. Christopher, H.J., Boardman, G.D., Freedman, D.L., 2000. Aerobic biological treatment of 2,4-dinitrotoluene in munitions plant wastewater. Water Res. 34, 1595e1603. Clark, B., Boopathy, R., 2007. Evaluation of bioremediation methods for the treatment of soil contaminated with explosives in Louisiana Army Ammunition Plant, Minden, Louisiana. J. Hazard. Mater. 143, 643e648. de Kreuk, M.K., Heijnen, J.J., van Loosdrecht, M.C.M., 2005. Simultaneous COD, nitrogen, and phosphorus removal by aerobic granular sludge. Biotechnol. Bioeng. 90, 761e769. Dodard, S.G., Renoux, A.Y., Hawari, J., Ampleman, G., Thiboutot, S., Sunahara, G.I., 1999. Ecotoxicity characterization of dinitrotoluenes and some of their reduced metabolites. Chemosphere 38, 2071e2079. Freedman, D.L., Shanley, R.S., Scholze, R.J., 1996. Aerobic biodegradation of 2,4dinitrotoluene, aminonitrotoluene isomers, and 2,4-diaminotoluene. J. Hazard. Mater. 49, 1e14. Gumuscu, B., Tekinay, T., 2013. Effective biodegradation of 2,4,6-trinitrotoluene using a novel bacterial strain isolated from TNT-contaminated soil. Int. Biodeterior. Biodegrad. 85, 35e41. Huang, J., Ning, G., Li, F., Sheng, G.D., 2015. Biotransformation of 2,4-dinitrotoluene by obligate marine Shewanella marisflavi EP1 under anaerobic conditions. Bioresour. Technol. 180, 200e206. Hughes, J.B., Wang, C.Y., Zhang, C., 1999. Anaerobic biotransformation of 2,4dinitrotoluene and 2,6-dinitrotoluene by Clostridium acetobutylicum: a pathway through dihydroxylamino intermediates. Environ. Sci. Technol. 33, 1065e1070. Khan, M.Z., Mondal, P.K., Sabir, S., 2011. Bioremediation of 2-chlorophenol containing wastewater by aerobic granules-kinetics and toxicity. J. Hazard. Mater. 190, 222e228. Kiran Kumar Reddy, G., Nancharaiah, Y.V., Venugopalan, V.P., 2014. Biodegradation of dibutyl phosphite by Sphingobium sp. AMGD5 isolated from aerobic granular biomass. Int. Biodeterior. Biodegrad. 91, 60e65. Kolekar, Y.M., Nemade, H.N., Markad, V.L., Adav, S.S., Patole, M.S., Kodam, K.M., 2012. Decolorization and biodegradation of azo dye, reactive blue 59 by aerobic granules. Bioresour. Technol. 104, 818e822. Küce, P., Coral, G., Kantar, Ç., 2015. Biodegradation of 2,4-dinitrotoluene (DNT) by Arthrobacter sp. K1 isolated from a crude oil contaminated soil. Ann. Microbiol.

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