Reduction of NOx in Fe-EDTA and Fe-NTA solutions by an enriched bacterial population

Bioresource Technology 130 (2013) 644–651

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Reduction of NOx in Fe-EDTA and Fe-NTA solutions by an enriched bacterial population B. Chandrashekhar, Padmaraj Pai, Amruta Morone, Nidhi Sahu, R.A. Pandey ⇑ Environmental Biotechnology Division, National Environmental Engineering Research Institute, Nehru Marg, Nagpur 440020, India

h i g h l i g h t s " An enriched biomass with Streptomyces sp., Enterobcater sp. and Citrobacter sp. was developed. III

" The biomass utilized NOx & Fe EDTA/NTA as terminal e



acceptors simultaneously.

" Being an electron donor, ethanol concentration affected the NOx reduction rates. II

II

" Both Fe NTA and Fe EDTA enhanced the NOx reduction rates using the biomass. " Higher growth and NOx reduction rates achieved in Fe-NTA media than Fe-EDTA media.

a r t i c l e

i n f o

Article history: Received 3 October 2012 Received in revised form 7 December 2012 Accepted 8 December 2012 Available online 20 December 2012 Keywords: Iron chelate Nitrogen oxide Anaerobes Growth kinetics Electron donor

a b s t r a c t An enriched biomass was developed from municipal sewage sludge consisting of three dominant bacteria, representing the genera of Enterobacter, Citrobacter and Streptomyces. The biomass was used in a series of batch experiments in order to determine kinetic constants associated with biomass growth and NOx reduction in aqueous Ferrous EDTA/NTA solutions and Ferric EDTA/NTA solutions using ethanol as organic electron donor. The maximum specific reduction rates of NOx in Ferrous EDTA and Ferrous NTA solutions were 0.037 and 0.047 mMoles L1 d1 mg1 biomass, respectively while in Ferric EDTA and Ferric NTA solutions were 0.022 and 0.024 mMoles L1 d1 mg1 biomass, respectively. In case of Ferric EDTA/NTA solution, the kinetic constants associated with reduction of Ferric EDTA/NTA to Ferrous EDTA/NTA were also evaluated simultaneously. The maximum specific reduction rates of Ferric EDTA and Ferric NTA were 0.0021 and 0.0026 mMoles L1 d1 mg1 biomass. The significance of these observations are presented and discussed in this paper. Ó 2012 Elsevier Ltd. All rights reserved.

1. Introduction Various adducts of chelating agents or ligands and metals viz. Ferrous (FeII) EDTA (ethylene diamine tetra acetic acid), Ferrous NTA (nitrilotriacetic acid) and Ferrous DTPA (diethylene triamine pentaacetic acid) have been used to enhance the mass transfer rate of NOx from gas phase to liquid phase in wet scrubbers by the formation of a stable Ferrous (Ligand)–NO complex (Gambardella et al., 2006; Demmink et al., 1997). FeIIEDTA and FeIINTA are the most commonly used chelates for absorption of NOx. In order to make absorption process economically feasible, the spent metal–ligand solution is regenerated and recycled by the process of biological denitrification in which the NO adduct of metal chelate is reduced to molecular nitrogen. Reduction of NO in such adducts is biologically catalyzed by using potential denitrifying bacterial ⇑ Corresponding author. Tel.: +91 712 2240097; fax: +91 712 2249900. E-mail address: [email protected] (R.A. Pandey). 0960-8524/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.biortech.2012.12.051

strains viz. Bacillus azotoformans (Kumaraswamy et al., 2005), Pseudomonas (Zhang et al., 2007), Paracoccus denitrificans (Li et al., 2012; Dong et al., 2012) and anaerobic sludge (Van der Maas et al., 2008; Dilmore, 2004) which use organic electron donor (e.g. ethanol) as the reducing agent. The regenerated adduct solution can be recycled back to the scrubber in a closed loop process where it continuously absorbs NOx from the emission gases. In order to optimize and design an engineered process for biological NOx removal using FeII (Ligand) and reducing bacteria, it is necessary to evaluate the kinetic parameters of bacterial growth and simultaneous NOx reduction under the operating conditions. In the present investigation, an enriched biomass was developed from municipal sewage sludge and evaluated for NOx reduction in FeIIEDTA & FeIIIEDTA (together denoted as Fe-EDTA) and in FeIINTA & FeIIINTA (together denoted as Fe-NTA) solutions using ethanol as carbon source as well as primary electron donor. A slow reduction of FeIIIEDTA and FeIIINTA to FeIIEDTA and FeIINTA respectively with the help of the enriched biomass has also been

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investigated. The kinetics associated with these phenomena are evaluated with the help of Monod kinetics equations and presented and discussed in this paper. 2. Methods 2.1. Source of biomass, enrichment and identification of bacteria The microorganisms used for this study were isolated from the sediment sludge of a sewage carrying canal. Enrichment of the biomass containing sludge was done in a bench scale unit and operated in batch mode. The unit consisted of a column packed with gravels (approximately 1 cm diameter) with a working volume of 500 mL and a feed reservoir. The feed reservoir was filled with 1 L of a medium containing 0.020 M FeIIEDTA with nutrients and trace elements. Nitrite (as NaNO2) was added to the medium along with ethanol and trace element solution from a concentrated feed stock whenever required. The unit was operated in anaerobic conditions in recirculation mode to allow bacterial growth in the packed column. The temperature of unit was controlled at 40 °C with the help of a water jacket. The enriched biomass collected from the unit after a period of 30–35 days was centrifuged (6000 rpm, 10 min) to harvest the mixed culture. The mixed culture was maintained in nutrient broth medium (HiMediaÒ) for further experiments. To isolate bacteria, the biomass was diluted in sterile distilled water and the bacteria were screened by growing on following types of solid media for 48 h: Nutrient agar medium (HiMediaÒ), Anaerobic agar medium (Accumix™) and Anaerobic agar medium supplemented with 5 mM Fe-EDTA. Bacterial colonies of different morphologies were selected and the strains were identified by16 sRNA amplification and sequencing followed by sequence similarity searches with BLAST program and phylogenetic analysis. The enriched biomass was used for growth and denitrification experiments in Fe-EDTA and Fe-NTA media separately by keeping all the environmental, nutritional and inoculum size uniform. 2.2. Media composition and batch experiments All the batch experiments for biomass growth and NOx reduction were conducted in 250 mL Erlenmeyer flasks containing 200 mL media. The flasks were sealed with silicon stoppers and maintained at 40 degrees C in a shaking incubator at 100 rpm. The experiments were conducted in order to find out the rate at which the nitrite was reduced by the biomass in Fe-EDTA and Fe-NTA at different initial ethanol concentrations, assuming that the growth and reduction rates would be proportional to the ethanol concentration. Hence, the assays were conducted at varying ethanol concentration (3–15 mM) in the media. Normal nitrite medium constituted: NaNO2 – (5 mM), K2HPO4 – (3 mM), KH2PO4 – (4 mM), MgCl2 – (0.002 mM), MgSO4.7H2O – (0.4 mM), Na2SO3 – (0.5 mM), FeSO4 – (0.06 mM), CuSO4.5H2O – (0.03 mM), and Na2MoO4 – (0.02 mM). FeIIEDTA–nitrite or FeIINTA–nitrite media were prepared by adding equimolar (20 mM) concentrations of FeCl2 and Na2H2EDTA or Na2NTA in a ratio of 1:1.2 to the nitrite medium under anoxic conditions. 10 mM FeIIIEDTA–nitrite and FeIIINTA–nitrite media were prepared by adding required amount of FeCl3 instead of FeCl2. The media as well as head space was flushed with pure nitrogen gas in order to maintain anaerobic conditions. The pH of the media was adjusted to 6.5 by adding NaOH or HCl for all the experiments. The biomass was inoculated to the medium at initial dry cell weight (DCW) concentration of about 80 mg L1. A medium inoculated with the biomass and without ethanol was kept as control for each experiment. The cumulative biomass concentration along with NO2 remaining in the media at various time

intervals was monitored in normal nitrite, FeIIIEDTA–nitrite and FeIIINTA–nitrite medium. Along with this, concentration of Fe3+ in the media was also monitored. NO2 reacts with FeIIEDTA and FeIINTA to form FeIIEDTA–NO and FeIINTA–NO adducts respectively, hence instead of NO2, concentration of NO remaining at various time intervals was monitored in these media. 2.3. Theory and calculations All the experiments were carried out to find out the effect of ethanol (carbon source) on growth rate, a correlation between log of biomass (lnx) and time (t) was made to calculate specific growth rate (l) at different initial concentration of ethanol (S) by using the Monod equation (Metcalf and Eddy, 2003)

l ¼ ðlnx2  lnx1 Þ=ðt2  t1 Þ

ð1Þ

Where, x1 and x2 are the biomass concentrations at time t1 and t2, respectively. It was found that l varies with (S), hence, the parameters of growth kinetics were evaluated from the double-reciprocal plots drawn between inverse of l and inverse of (S) (Metcalf and Eddy, 2003) which gives a relationship between lmax and KS as shown below

1=l ¼ K s =½lmax  ðSÞ þ 1=lmax

ð2Þ

where, lmax is the maximum specific growth rate and Ks is the half saturation constant for ethanol. Eq. (1) for calculating growth rates could be modified and used for calculating the specific NO2 reduction rate (V NO2 ) and specific NO reduction rate (VNO) simultaneously occurring in the media as shown below

V NO2 orV NO ¼ ðlnN2  lnN1 Þ=ðt2  t 1 Þ  x

ð3Þ III

where, N1 and N2 are the concentration in Fe EDTA and FeIIINTA media at time t1 and t2, respectively. In case of FeIIEDTA and FeIINTA media, N1 and N2 denote the FeII (Ligand)–NO concentrations at time t1 and t2, respectively. x is the initial biomass concentration in both the cases. Similarly, the above equation was also used to calculate the specific Fe3+ reduction rate (VFe3+) in FeIIIEDTA and FeIIINTA media as shown below NO2

V Fe3þ ¼ ðlnf2  lnf1 Þ=ðt2  t 1 Þ  x

ð4Þ

3+

III

III

where, f1 and f2 are the Fe concentration in Fe EDTA and Fe NTA media at time t1 and t2, respectively and x is the initial biomass concentration. The specific reduction rates (V) calculated by the above equations also varied according to initial ethanol concentration. Hence, values of maximum specific reduction rate, Vmax and half saturation constant, KR for ethanol were evaluated from the double-reciprocal plots drawn between inverse of (V) and inverse of (S), which gave a relationship between Vmax and KR as shown below:

1=V ¼ K R =ðV max  SÞ þ 1=V max

ð5Þ NO2

In this paper, Vmax is written as V for maximum specific nitrite reduction rate in normal nitrite, FeIIIEDTA–nitrite and FeIIINTA–nitrite media, as VmaxNO for maximum specific NO reduction rate in FeIIEDTA and FeIINTA media and as VmaxFe3+ for maximum specific Fe3+ reduction rate in FeIIIEDTA–nitrite/NTA–nitrite media, respectively. 2.4. Analytical methods NO2 concentration was estimated by a standard method (APHA, 2005). Equimolar solutions of FeIIEDTA and FeIINTA coupled

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to an NO adduct showed peak absorbances at 435 and 423 nm, respectively using a spectrophotometer (Shimadzu UV-1800). Hence, the concentration of FeIIEDTA–NO and FeIINTA–NO was determined by directly measuring their absorbance (Schneppensieper et al., 2001). Biomass was removed from the media by using 0.22 lm PVDF membrane filter (Millex GV, Millipore, Ireland) before measuring NO concentration. Total Fe and Fe2+ (in the form of FeIIEDTA or FeIINTA) ions were estimated by a method using Ferrozine reagent (Viollier et al., 2000). Fe3+ concentration (in the form of FeIIIEDTA or FeIIINTA) was calculated as the difference between total iron and Fe2+ ions. Ethanol concentration remaining at the end of experiments was estimated by a miniaturized form of potassium dichromate method described by Caputi et al. (1968). Cell density of homogenized culture was determined by measuring optical density of the culture at wavelength of 600 nm (OD600) with a UV spectrophotometer. Then OD600 results were converted to dry cell weight by a correlation obtained from a calibration curve, which was developed by plotting dry weight of biomass (X) per liter against OD600 (1 OD600 = 3.18 Xg/L).

3. Results and discussion 3.1. Enrichment of biomass, isolation and identification of bacteria Enrichment of the sludge containing biomass was carried out in a bench scale unit, operated under anoxic conditions with 0.020 M Fe-EDTA medium in closed-loop recirculation mode using ethanol as carbon source. Initially when nitrite was not added, Fe2+ concentration in the medium increased from 0.014 to 0.016 M during the first five days of operation as shown in Fig. 1A. On the sixth day, when 1 mM nitrite was added to the medium, there was a decrease in Fe2+ concentration in the medium suggesting oxidation of FeIIEDTA to FeIIIEDTA by NO2. This also led to reduction of NO2 to NO and formation of FeIIEDTA–NO complex in the medium. NO was completely reduced by the biomass along with partial reduction of FeIIIEDTA to FeIIEDTA during the next 3 days. This process was repeated for another 22 days during which similar series of oxidation–reduction reactions took place at various initial NO2 concentrations as shown in Fig. 1A. On 30th day of operation, the medium contained 0.0175 mM FeIIEDTA and about 0.1 mM NO. The continuous reduction of NO adduct of FeIIEDTA indicated that the biomass was enriched with bacteria capable of reducing NO complexed with FeIIEDTA. An increase in Fe2+ concentration in the medium indicated the iron reduction potential in the enriched biomass. The enriched biomass spread on nutrient agar & anaerobic agar media produced numerous colonies of bacteria. Three morphologically distinct and most common colony types (named as A1-EBD, A2-EBD and A3-EBD) were identified in the colonies obtained in these media. But in anaerobic agar medium supplemented with Fe-EDTA, only one type of colony (A2-EBD) was observed. Using colony count method to enumerate the colony types, it was found that A1-EBD and A3-EBD represented more than 90% of total population of bacteria in both nutrient agar and anaerobic agar media at a dilution factor of 102 while A2-EBD dominated (92%) in anaerobic agar medium supplemented with Fe-EDTA at the same dilution. The 16s rDNA sequences of A1-EBD, A2-EBD and A3EBD showed a percentage identity of more than 99% with Enterobacter sp., Streptomyces sp. and Citrobacter sp., respectively. Based on 16S rRNA nucleotide homology and phylogenetic analysis (Fig. 1B) the three strains were identified as follows: A1-EBDEnterobacter amnigenus, A2-EBD- Streptomyces caelestis and A3EBD- Citrobacter fruendii. NOx reduction ability of Enterobacter sp., Citrobacter sp., and Streptomyces sp., has been widely reported previously. The capabil-

ity for NO3 respiration is found in several genera of soil facultative anaerobes such as Citrobacter, Enterobacter, Erwinia, Escherichia, and Klebsiella (Tiedje, 1988). Zumft (1997) has reported that members of Enterobacteriacea species are not denitrifiers, but capable of dissimilatory nitrate reduction to ammonia. Fazzolari et al. (1990) has reported the dissimilatory reduction of nitrate and nitrite to nitrous oxide and ammonia (but not nitrogen) by E. amnigenus. Enterobacter cloacae have been reported to perform reduction of FeIIEDTA–NO to produce N2 using glucose as electron donor (Zhang et al., 2008). Dissimilatory reduction of NO2 to N2O and NH4+ has also been reported by using Citrobacter sp. In a batch culture, a soil Citrobacter sp. produced N2O and NH4+ by enzymatically reducing NO2 (Smith, 1982). Nitrate and nitrite reduction by Citrobacter diversus under aerobic environment has been reported by Huang and Tseng (2001) which could reduce about 95% of 16.5 mg L1NO2 to N2 within 20 h. Kumon et al. (2002) have screened and identified actinomycetes strains and found that the facultative anaerobe Streptomyces antibioticus evolves N2 and some N2O from nitrate (NO3) and that the cell growth was dependent on nitrate respiration. Hence, it can be suggested that in the enriched biomass used in the present work, E. amnigenus and C. fruendii is responsible for reduction of NO2 to NH4+ via NO and N2O while, S. caelestis could be carrying out denitrification of NO2 and NO to N2. Since, S. caelestis is identified as the most dominant species in the bacterial consortium in the presence of Fe-EDTA, most of the NOx reduction would be carried out by this species. However, the present investigation focuses on overall reduction of NOx (NO2 and NO) in aqueous Fe-EDTA and Fe-NTA solutions by the enriched biomass without characterizing the end-products of reduction. 3.2. Biomass growth and nitrite reduction in normal nitrite, Ferric EDTA–nitrite and Ferric NTA–nitrite media The results of biomass growth and nitrite reduction profiles are shown in Fig. 2A–C. In all the cases, a distinct lag phase was observed during the biomass cultivation. The lag phase seemed to be longer in FeIIIEDTA–nitrite and FeIIINTA–nitrite medium than in normal nitrite medium. The biomass yield (Y) obtained at 15 mM initial ethanol concentration was 53.69, 33.0 and 35.5 mg DCW mM1ethanol L1 in normal nitrite, FeIIIEDTA–nitrite and FeIIINTA–nitrite media. The specific growth rate (l) calculated by Eq. (1) increased with initial ethanol concentration (S) in each of the three media and the growth of biomass occurred at different rates in different media as shown in Fig. 3A. Using a double-reciprocal plot [1/l vs. 1/S] and Eq. (2), lmax and KS were calculated for each media as shown in Table 1. As expected, it was observed that lmax was higher (0.044 h) in normal nitrite medium than either FeIIIEDTA–nitrite or FeIIINTA–nitrite medium. This indicated that, addition of either FeIIIEDTA or FeIIINTA to the media drastically reduced the biomass growth rate, which is inconsistent with the observation by Zhang et al., 2009 where growth rate of Escherichia coli was enhanced by FeIIIEDTA in the presence of NOx. A marginal difference between the values of lmax obtained in FeIIIEDTA–nitrite and FeIIINTA–nitrite media (0.030 and 0.034 h1, respectively) indicated the biomass could grow at almost similar rates, however, a less KS value in FeIIIEDTA–nitrite medium also indicated that ethanol consumption was lesser in this medium, as compared to FeIIINTA–nitrite medium. A decline in concentration of NO2 was observed alongside biomass growth in all the media. In normal nitrite and FeIIIEDTA– nitrite medium, the decline rate was slower during the lag phase, and sharper during the log phase, when biomass concentration increased rapidly and after that remained almost constant (Fig. 2A–B). Similar phenomenon was observed in FeIIINTA–nitrite medium, but however, the decline occurred at similar rates during

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647

A

B

Fig. 1. (A). Enrichment of biomass showing reduction of NO adduct of FeIIEDTA and generation of Fe2+ ions as a result of iron reduction (B). Phylograms of isolated bacteria viz. A1-EBD, A2-EBD and A3-EBD from the enriched biomass.

lag and log phases (Fig. 2C). These results indicated that nitrite reduction was more related to biomass growth in normal nitrite and FeIIIEDTA–nitrite medium than in FeIINTA–nitrite medium. Like growth rates, the NO2 reduction rates in each media can also be related to initial ethanol concentration, which acts as an electron donor. The specific nitrite reduction rate (V NO2 ) calculated by Eq. (3) increased with increasing initial ethanol concentration (S) in each of the three media and also, the reduction occurred at different rates in the media as shown in Fig. 3B. Using a double reciprocal plot [1/V NO2 and 1/S] and Eq. (4), V max NO2 was estimated in each of the media as shown in Table 1. It was found that V max NO2 was almost equal in FeIIIEDTA–nitrite and FeIIINTA–nitrite media while the KR was considerably higher in FeIIINTA medium than in FeIIIEDTA media. Moreover, V max NO2 in normal nitrite medium was found to be slightly lesser than that in FeIIIEDTA and FeIIINTA media even when the growth was significantly reduced in FeIIIEDTA and FeIIINTA media. This indicated that FeIIIEDTA or FeIIINTA also could slightly increase the nitrite reduction rate. Zhang et al., 2009 also reported that the reduction rate of NO2 or NO was

enhanced in presence of FeIIIEDTA. The reason behind this phenomenon could be the simultaneous iron reduction taking place in the media, which will be discussed later. 3.3. Biomass growth and nitrite reduction in FeIIEDTA–nitrite and FeIINTA–nitrite media The results of biomass growth profiles are shown in Fig. 4A and B. In this case, no distinct lag phase was observed as compared to FeIIIEDTA–nitrite and FeIIINTA–nitrite media. The biomass yield (Y) obtained at 15 mM initial ethanol concentration was 38.45 and 39.12 mg DCW (mM Ethanol)1 L1 in FeIIEDTA–nitrite and FeIINTA–nitrite media, respectively. The specific growth rate (l) calculated by Eq. (1) increased with initial ethanol concentration (S) in each of these media and the growth of biomass occurred at different rates in different media as shown in Fig. 5A. Using a double reciprocal plot [1/l vs 1/S] and Eq. (2), lmax and KS were calculated for each media as shown in Table 1. It was observed that lmax in either FeIIEDTA–nitrite or FeIINTA–nitrite medium was lesser than

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A

A

B

B

C

Fig. 2. Profiles of biomass growth and NO2 concentration in (A). normal nitrite medium (B). FeIIIEDTA–nitrite medium and (C). FeIIINTA–nitrite medium at 15 mM initial ethanol concentration.

in normal nitrite medium (0.044 h1) with lmax in FeIINTA–nitrite medium (0.038 h1) being higher than to FeIIEDTA–nitrite medium (0.034 h1). The value of KS for ethanol was higher in case of FeIINTA–nitrite medium. KS for growth was also higher in FeIIINTA–nitrite medium than in FeIIIEDTA–nitrite medium. These results indicate that biomass growth in Fe-NTA medium consumed more ethanol than Fe-EDTA medium for almost similar lmax values. Li et al. (2007) also observed that the E. coli cell growth rate decreased in the presence of FeIIEDTA and FeIIEDTA–NO and no cell growth was observed at FeIIEDTA–NO concentration at 3.7 mM and at FeIIEDTA concentration of 8.8 mM. Similar results were found in this investigation as lmax decreased when FeIIEDTA or FeIINTA was added to normal nitrite media. However the biomass could grow at higher FeIIEDTA and FeIINTA concentration

Fig. 3. Effect of initial ethanol concentration (3–15 mM) on (A). Specific biomass growth rate (B). Specific NO2 reduction rate (V NO2 ) & specific Fe3+ reduction rate in FeIIIEDTA–nitrite and FeIIINTA–nitrite media.

(20 mM) and higher NOx concentration (5 mM). Hence, the biomass used in our study could grow at higher concentration of EDTA & NTA and higher concentration of NOx than E.Coli. Looking at the biomass yields in different media for NOx reduction (Table 1), FeNTA medium yields more biomass than Fe-EDTA media. A higher bacterial yield was also obtained in the presence of NTA as compared to EDTA (Yuan and VanBriesen, 2008). Before adding the inoculum for conducting growth and NOx reduction experiments, 20 mM FeIIEDTA and FeIINTA was added to 5 mM nitrite media to prepare FeIIEDTA–nitrite and FeIIINTA–nitrite media respectively. But, this decreased the concentration of free NO2, leaving minimum residual NO2 concentration in the media. This was also indicated by a rapid change in the color of media from colorless to brownish green, which indicated the formation of FeIIEDTA–NO2 and FeIINTA–NO2 complex in the media, which can be represented by the following equations:

2FeII EDTA2 þ NO2 þ 2Hþ ! FeII EDTA-NO2 þ FeIII EDTA þ H2 O 2FeII NTA2 þ NO2 þ 2Hþ ! FeII NTA-NO2 þ FeIII NTA þ H2 O

ð6Þ ð7Þ

Hence, in these media one of the steps involved during reduction of NO2 to N2 (i.e. NO2 ? NO ? N2) was accomplished instantaneously. Going by the stoichiometry of the above equations, NO2 oxidizes equimolar concentrations of FeIIEDTA or FeIINTA to yield equimolar concentrations of FeIIIEDTA or FeIINTA, respectively. The NO formed as a result of this reaction complex with the remaining FeIIEDTA or FeIINTA. An oxygen depleted environment is thus developed within the media which is favorable for

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B. Chandrashekhar et al. / Bioresource Technology 130 (2013) 644–651 Table 1 Parameters of growth kinetics and NOx reduction kinetics in different media. Media

Growth kinetics parameters 1

lmax h

a b c

Biomass yield (Y) mg DCW mM1 L1

Reduction kinetics parameters Vmax mMoles L1 d1 mg1 DCW

Ks mM ethanol

Normal Nitrite Medium FeIIEDTA–nitrite Medium FeIINTA–nitrite Medium FeIIIEDTA–nitrite Medium

0.044 0.034 0.038 0.030

2.10 1.40 2.30 0.84

53.69 38.45 39.12 33.00

FeIIINTA–nitrite Medium

0.034

1.71

35.50

KR mM ethanol

NOx Reduction

Fe3+ Reduction

0.019a 0.037b 0.047b 0.022a – 0.024a –

– – – – 0.0021c – 0.0026c

3.35 9.47 10.95 3.64 1.82 4.42 2.12

Maximum specific NO2 reduction rate, V max NO2 . Maximum specific NO reduction rate, VmaxNO. Maximum specific Fe3+ reduction rate, VmaxFe3+.

A

Fig. 5. Effect of initial ethanol concentration (5–15 mM) on specific biomass growth rate and specific NO reduction rate (VNO) in FeIIEDTA and FeIINTA media.

B

Table 2 Characteristics of nitrite media after addition of FeIIEDTA and FeIINTA.

Fig. 4. Profiles of biomass growth, NO and Fe2+ concentration in (A). FeIIEDTA– nitrite medium and (B). FeIINTA–nitrite medium at 15 mM initial ethanol concentration. The figures show slight increase in Fe2+ concentration (after an initial decline) after most of NO is reduced.

NO reduction. Table 2 shows the pH and concentrations of FeIIEDTA/NTA, FeIIIEDTA/NTA and FeIIEDTA/NTA–NO2 in the media immediately after addition of FeIIEDTA and FeIINTA under anoxic conditions. After the inoculum and incubation, a decline in concentration of NO was also observed simultaneously during the growth of biomass in the media as shown in Fig. 4A and B. During this reduction process, the pH gradually dropped to below 5 from an initial value of 6.78 which may be due to the release of H+ ions according to the following equation:

6FeII EDTA-NO2 þ C2 H5 OH ! 2HCO3 þ 2Hþ þ 3N2 þ H2 O þ 6FeII EDTA2

ð8Þ

Characteristic

FeIIEDTA–nitrite media

FeIINTA–nitrite media

Total Fe Initial Nitrite Mean FeIIEDTA/NTA–NO Mean FeIIIEDTA/NTA Mean pH

20 mM 5 mM 4.5 mM 6.2 mM 6.78

20 mM 5 mM 4.5 mM 6.6 mM 6.84

The specific NO reduction rate (VNO) calculated by Eq. (3) increased with initial ethanol concentration (S) in each of the media and reduction occurred at different rates in different media as shown in Fig. 5. Using a double reciprocal plot [1/VNO and 1/S] and Eq. (4), VmaxNO and KR were estimated in both media as shown in Table 1. The maximum specific NO reduction rate, VmaxNO in FeIINTA medium was found to be higher (0.047 mMoles L1 mg DCW1 day1) than in FeIIEDTA medium (0.037 mMoles L1 mg DCW1 day1). A higher NO reduction rate in FeIINTA–NO medium can be justified by various reasons. First, the stability constants of FeIIEDTA–NO is higher than FeIINTA–NO complex, 2.1  106 and 1.8  106, respectively (Wolak and Eldik, 2002). Further, the stability constant of FeIIEDTA alone is also higher than FeIINTA (Martell and Smith, 1974; IUPAC, 1982). Hence, being more stable, FeIIEDTA–NO complex would make the availability of NO for enzyme mediated reduction more difficult, resulting in lower NO reduction rate as compared to FeIINTA medium. Second, higher VmaxNO in FeIINTA medium could also be attributed to higher lmax and YX/S in FeIINTA–nitrite medium as compared to FeIIEDTA–nitrite medium. Third, nitrate and nitrite reduction mediated by

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NTA itself has been reported during which NTA acts as reducing agent and subsequently gets oxidized and degraded (Wanner and Egli, 1993). This mechanism could also be responsible for the higher NOx reduction rate in FeIINTA medium. However, such mechanism is not yet reported in the presence of EDTA. Interestingly, the maximum specific nitrite reduction rate, V max NO2 V max NO2 in normal nitrite medium was much lesser than VmaxNO in the media having either FeIIEDTA or FeIINTA even when lmax in normal nitrite media was considerably higher (Table 1). Recent report has shown that presence of Fe2+ ions inhibits the rate of NOx reduction by formation of ferric hydroxide coating on the cell via a surface-catalyzed, abiotic reaction between Fe2+ and NOx. This coating inhibits reduction of NOx by physically blocking transport into the cell (Cooper et al., 2003). Using P. denitrificans, Coby and Picardal, 2005 showed that the presence of Fe2+ on the surface of cells leads to the inhibition of NO reduction even at small concentrations of Fe2+ ions. However, in the present investigation using the enriched biomass, Fe2+ did not show any inhibitory effect on NOx reduction; rather, the rate of NOx reduction was increased in the presence of Fe2+ ions. This could be due to two reasonsfirst, the powerful chelating ability of NTA and EDTA which might prevent the abiotic reaction between Fe2+ and NO2 ions on the cell surface, and second, the electron donating potential of FeIIEDTA and FeIINTA complexes. Ethanol and other organic substances are known to be excellent electron donors for denitrification and is also reported to be the primary electron donor for denitrification in the presence of FeIIEDTA adducts (Li et al., 2012; Zhang et al., 2007; Dilmore et al., 2007) along with other organic substances such as such as glucose, methanol, lactate and acetate (Dong et al., 2012; Li et al., 2007). Using glucose as the electron donor and E. cloacae as the microorganism, Zhang et al. (2008) obtained a maximum reduction capacity of 0.818 mM h1. Ethanol acts as a primary electron donor for reducing both FeIIEDTA–NO and FeIIIEDTA in the BioDeNOx process (Dilmore et al., 2007). However, there is an ongoing debate on the primary electron donor as FeIIEDTA has also been reported as an electron donor by different researchers in the past, though it is not an energetically favorable electron donor for NOx reduction as compared to ethanol. This also results in oxidation of FeIIEDTA to FeIIIEDTA (Hauck et al., 2001; Van der Maas et al., 2005; Kumaraswamy et al., 2005). Van der Maas et al., (2008) have evaluated the reduction rate of FeIIEDTA–NO in 5–25 mM FeIIEDTA solution by using denitrifying sludge and BioDeNOx reactor mixed liquor and proved that ethanol was not responsible for reduction for NO rather, FeIIEDTA acted as the primary reducing agent. They achieved a specific NO reduction rate of 0.34 nmol (mg protein)1 s1 using biomass obtained from BioDeNOx liquor and reported that the redox system of FeIIEDTA/FeIIIEDTA in the media interferes with the NO reduction transfer chain and enhances the NO reduction rate and accordingly the following stoichiometric equations were put forward by them.

2FeII EDTA2 -NO þ 2Hþ ! N2 O þ H2 O þ 2FeIII EDTA

ð9Þ

N2 O þ FeII EDTA2 þ 2Hþ ! N2 þ H2 O þ 2FeIII EDTA

ð10Þ

In the present investigation, on increasing the initial ethanol concentration the specific NO reduction rate was increased, thus ethanol acted as electron donor. Further, VmaxNO was higher in the presence of FeIIEDTA and FeIINTA than V max NO2 which indicated that FeIINTA also possesses electron donating ability similar to FeIIEDTA and hence FeIIEDTA or FeIINTA and ethanol contributed to the overall NO reduction rates. 3.4. Reduction of FeIIIEDTA/NTA to FeIIEDTA/NTA The effectiveness of BioDeNOx process utilizing either FeIIEDTA or FeIINTA largely depends on how effectively the biomass is able

to reduce oxidized FeIIIEDTA or FeIIINTA because much of the iron gets oxidized by the presence of oxygen and NO2 in the gas according to Eqs. (6) and (7). It is also proved that FeIIEDTA acts as electron donors for NO reduction, resulting in decrease in concentration of FeIIEDTA in the medium due to its conversion to FeIIIEDTA according to Eqs. (9) and (10). To make the scrubbing liquid repeatedly recyclable in the process, it is necessary to prevent oxidation of iron which can be accomplished by simultaneous biological reduction of iron with the help of biomass which takes place according to the following equation using ethanol as electron donor.

12FeIII EDTA þ C2 H5 OH þ 5H2 O ! 2HCO3 þ 12FeII EDTA2 þ 14Hþ

ð11Þ

During enrichment period, both FeIIEDTA2 and FeIIIEDTA existed in the medium and under denitrifying conditions the biomass showed an ability to reduce iron. A slight increase in FeIIEDTA2 and FeIINTA2 concentration (after an initial decline phase) was also observed during the batch growth and reduction experiments in FeIIEDTA–nitrite and FeIINTA–nitrite media (Fig. 4A and B) showing that the reduction potential in the media persists even after almost complete reduction of NO in FeIIEDTA or FeIIEDTA medium. Therefore, there is a partial reduction of FeIIIEDTA and FeIIINTA to their respective FeIIEDTA and FeIINTA forms with the help of remaining ethanol in the media. During reduction of NO2 in 10 mM FeIIIEDTA and FeIIINTA media under anaerobic conditions, a decline in Fe3+ concentration was also observed indicating iron reduction and its conversion to Fe2+ (Fig. 2B and C). Similar to nitrite reduction, the specific iron reduction rate, VFe3+ as calculated by Eq. (4) increased with initial ethanol concentration (S) in each of the media as shown in Fig. 3B. Using a double reciprocal plot [1/VFe3+ vs 1/S] and Eq. (5), VmaxFe3+ was calculated for each media as shown in Table 1. A higher VmaxFe3+ was observed in FeIIINTA medium (0.0021 mMoles L1 d1 mg1 DCW) as compared to FeIIIEDTA medium (0.0026 mMoles L1 d1 mg1 DCW) though the difference was marginal. At an initial ethanol concentration of 15 mM, the final Fe3+ reduction efficiency reached about 31% and 35% in FeIIIEDTA–nitrite and FeIIINTA–nitrite medium respectively at t = 54 h. The reduction obtained was very slow as compared to that obtained by Enterococcus sp. which reduced 63.2% of 10 mM FeIICitrate at t = 50 h at a rate of 0.21 mM h1 in the presence of 8 mM NO2 and glucose as electron donor (Li et al., 2011). P. denitrificans could reduce 92% of 12 mM FeIIIEDTA at t = 6 h at a rate of 0.14 mM h1 using ethanol as electron donor (Dong et al., 2012). However, both nitrite and FeIIIEDTA or FeIIINTA reduction was achieved simultaneously at an almost constant rate throughout the experiment with a total reduction of more than 3 mM Fe3+ ions in 54 h. In contrast, Gao et al. (2011) also achieved a reduction of 3.31 mM FeIIIEDTA by Pseudomonas sp., but the start of reduction was delayed by almost 25 h. Thus, the biomass used for the present study is a very slow, but constant reducer of FeIIIEDTA and FeIIINTA in the presence of nitrite. Slow Fe3+ reduction rate can be attributed to the presence of NO2 in the media. Generally, NOx reduction is preferred over iron reduction by such bacteria capable of catalyzing the reduction of both NOx and iron (Van der Maas et al., 2005; Dilmore, 2004; Kanso et al., 2002). Using a mixed culture containing Pseudomonas sp., Gao et al. (2011) have also proved that reduction of FeIIIEDTA and generation of FeIIEDTA started only after most of the NO had been reduced. However, Zhang et al. (2009) showed that the FeIIIEDTA reduction rate decreases with the increase of NO2 or NO3 addition, but both FeIIIEDTA and NO2 reduction can take place simultaneously by E. coli cells. Reduction of FeIIIEDTA should produce FeIIEDTA which reacts with available NO2 to form FeIIEDTA–NO, thus increasing NO2 reduction rates, which is also reported by Zhang et al. (2009). This is consistent with our observations as

B. Chandrashekhar et al. / Bioresource Technology 130 (2013) 644–651

presence of FeIIIEDTA and FeIIINTA slightly enhanced the maximum NO2 reduction rates (Table 1). Presence of FeIIEDTA–NO and FeIIEDTA itself also inhibits the reduction rate of FeIIIEDTA. Li et al. (2007) used E. coli to demonstrate that increasing FeIIEDTA–NO and FeIIEDTA in the system can inhibit both the FR-2 cell growth and thus affect the FeIIIEDTA reduction. Therefore, the Fe3+ reduction rates were higher during the earlier stages of batch experiments when FeIIEDTA was minimum. On comparing the nitrite reduction rates, V max NO2 in FeIIIEDTA and FeIIINTA media was slightly higher (0.022 and 0.024 mMoles L1 d1 mg1 DCW respectively) than that in normal nitrite medium (0.019 mMoles L1 d1 mg1 DCW). Since, VmaxFe3+ was higher in FeIIINTA– nitrite medium and higher reduction rate of iron would yield more Fe2+ in the medium, it could be the reason for a slightly higher NO2 reduction rate in FeIIINTA–nitrite medium as compared to FeIIIEDTA–nitrite medium. Based on the results obtained, a bioreactor utilizing FeIINTA as the NOx absorbing medium and the enriched biomass for reduction of absorbed NOx is under development phase. The main objective of operating the bioreactor is to optimize the simultaneous NOx and iron reduction abilities of the biomass at various NOx and Fe-NTA loading rates. Degradation of NTA that could enhance NOx as well as iron reduction is one of the key parameters. Since NTA is more biodegradable than EDTA, it could be used to replace more generally used EDTA in the media. 4. Conclusion The enriched biomass having S. caelestis, E. amnigenus and C. fruendii could utilize NOx and FeIIIEDTA/FeIIINTA as terminal electron acceptors simultaneously in Fe-EDTA and Fe-NTA media. Ethanol acted as electron donor in all the media, whose concentration affected the NOx and Fe3+ reduction rates. Addition of FeIIEDTA or FeIINTA in nitrite medium enhanced NOx reduction rates even at slower growth rates suggesting that FeIIEDTA and FeIINTA also acted as electron donors. On comparing the biomass yields, NOx and Fe3+ reduction rates, it is suggested that Fe-NTA is more compatible medium than Fe-EDTA for biological NOx reduction. Acknowledgements The authors are thankful to Director, NEERI, to give kind permission to publish this research work. The financial support extended by Department of Biotechnology and Council of Scientific and Industrial Research, Ministry of Science & Technology, Government of India, for execution of this project work is duly acknowledged. References American Public Health Association (APHA), 2005. Standard Methods for the Examination of Water and Wastewater, nineteenth ed. American Public Health Association, Washington, DC, USA. Caputi, A.J., Ueda, M., Brown, T., 1968. Spectrophotometric determination of ethanol in wine. Am. J. Enol. Vitic. 19, 160–165. Coby, A.J., Picardal, F.W., 2005. Inhibition of NO3 and NO reduction by microbial Fe(III) reduction: evidence of a reaction between NO2 and cell surface-bound Fe2+. Appl. Environ. Microbiol. 71 (9), 5267–5274. Cooper, D.C., Picardal, F.W., Schimmelmann, A., Coby, A.J., 2003. Chemical and biological interactions during nitrate and goethite reduction by Shewanella putrefaciens 200. Appl. Environ. Microbiol. 69, 3517–3525. Demmink, J.F., Van Gils, I.C.F., Beenackers, A.A., 1997. Absorption of nitric oxide into aqueous solutions of ferrous chelates accompanied by instantaneous reaction. Ind. Eng. Chem. Res. 36, 4914–4927. Dilmore, R., 2004. Evaluation of the kinetics of biologically catalyzed treatment and regeneration of NOx scrubbing process waters. Doctoral Thesis. University of Pittsburgh, 224pp. Dilmore, R., Neufeld, R.D., Hammack, R.W., 2007. Kinetics of chemoheterotrophic microbially mediated reduction of ferric EDTA and the nitrosyl adduct of ferrous

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