Nitritation–denitritation in landfill leachate with glycerine as a carbon source

Bioresource Technology 142 (2013) 297–303

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Nitritation–denitritation in landfill leachate with glycerine as a carbon source Dorota Kulikowska, Katarzyna Bernat ⇑ University of Warmia and Mazury in Olsztyn, Department of Environmental Biotechnology, Słoneczna Str. 45G, Olsztyn 10-709, Poland

h i g h l i g h t s  Nitrogen removal from municipal landfill leachate via nitritation–denitritation.  At limited oxygen concentration nitritation–denitritation occurred concurrently.  Glycerine (Gly) may be successfully used as carbon source for denitritation.  Gly increase in the total carbon source caused decrease in biomass production.

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Article history: Received 21 March 2013 Received in revised form 27 April 2013 Accepted 29 April 2013 Available online 9 May 2013 Keywords: Municipal landfill leachate SBR Nitritation–denitritation COD removal Sludge production

a b s t r a c t The effects of limited oxygen concentration (0.7 mg O2/L) in the aeration phase of the SBR cycle and glycerine as an additional carbon source on the effectiveness of nitritation–denitritation and sludge production during municipal landfill leachate treatment were examined. As carbon sources, sodium acetate (Ac) and sodium acetate (Ac) with glycerine (Gly) in the proportions of 3:1 (v/v) and 1:1 (v/v) were added. Low dissolved oxygen concentration inhibited the second stage of nitrification and nitrites were the main final products. Nitritation effectiveness was ca. 98–99%. Denitritation efficiency was relatively low (61%) in the reactor fed with Ac, which may be linked with high sludge production (Yobs – 0.6 mg VSS/mg COD). Glycerine addition (Ac:Gly 1:1, v/v) caused an increase in process efficiency to 75.6% with a concurrent significant decrease in biomass production (Yobs – 0.46 mg VSS/mg COD). Ó 2013 Elsevier Ltd. All rights reserved.

1. Introduction Landfill leachates are considered wastewater that is difficult to treat since they undergo continuous qualitative and quantitative changes with landfill aging. Leachates from stabilized landfill contains high levels of ammonium, often reaching several thousands of milligrams per liter (Liang and Liu, 2008; Zhu et al., 2013) and a low concentration of biodegradable organics. Therefore, biological nitrogen removal via denitrification, proceeded by nitrification, is hindered by a low C/N ratio and to obtain high process effectiveness an external carbon source is needed. To date, the most popular are commercially available carbon sources such as methanol, ethanol or acetic acid, although this generates additional treatment costs. In order to eliminate this problem, two differentiated approaches may be applied: (i) technological solutions involving processes based on partial nitrification (oxidation of ammonia nitrogen to nitrite; nitritation) in combination with a short-cut ⇑ Corresponding author. Tel.: +48 89 523 41 18; fax: +48 89 523 41 31. E-mail addresses: [email protected] (D. Kulikowska), katarzyna.bernat@ uwm.edu.pl (K. Bernat). 0960-8524/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.biortech.2013.04.119

denitrification (denitritation) (Fux et al., 2006; Aslan and Dahab, 2008) and (ii) the use of waste products as a potential carbon source for denitrification (Tora et al., 2011; Frison et al., 2013). In the presented experiment, it was proposed that nitrogen removal from municipal landfill leachate would be due to both above-mentioned approaches, i.e. nitritation–denitritation and glycerine – waste-product from biodiesel production as a carbon source. Partial nitrification requires a reduction in the activity of the nitrite oxidizing bacteria (NOB), without affecting the ammonia oxidizing microorganisms (AOB). One way to achieve this is to utilize the difference in the activation energies between ammonia oxidation (68 kJ/mol) and nitrite oxidation (44 kJ/mol). In the case of ammonia the higher activation energy means that the process rate can be made temperature-dependent (Schmidt et al., 2002). Further parameters conducive to short-cutting nitrification may be pH and dissolved oxygen (DO) regulation. However, in case of pH, discrepancies can be noted among the existing data (Villaverde et al., 1997; Ruiz et al., 2003; Wang et al., 2007). At low DO concentrations ammonia oxidizing bacteria are known to have a higher affinity for oxygen than nitrite oxidizing bacteria. Therefore the

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first phase of nitrification dominates (Schmidt et al., 2003; Zeng et al., 2009; Yang and Yang, 2011). So, in presented study, a control strategy to obtain nitritation was to adjust the air supply in the aeration phase to attain the required low dissolved oxygen concentration. Earlier studies concerning reject water treatment in SBR proved that this strategy is efficient for obtaining nitrite as a final nitrification product (Bernat et al., 2012, 2013). The possibility of nitrogen removal from wastewater with a low C/N ratio is currently a subject of interest to many researchers. However, most of the works focus only on the process efficiency (Xu et al., 2012). It is known that in wastewater treatment by activated sludge, new cells (sludge) are one of the final products. Currently, with increased restrictions in sludge reuse and disposal, sludge treatment has become more challenging and more costly. Thus, an ideal approach to the sludge problem would be to reduce the excess sludge in wastewater treatment rather than post-treat the produced sludge. To reduce the production of biomass, the wastewater process must be engineered to divert the substrate from assimilation for biosynthesis to fuel exothermic, non-growth activities. For example, Abbassi et al. (1999) showed that a reduction of the excess sludge production by about 25% can be achieved by raising the oxygen concentration from 2 to 6 mg O2/L in the mixed liquor. Partial nitrification is, on the other hand, observed at low DO concentration. Hence, an assessment of biomass production at low oxygen concentrations is needed. Similarly, it is important to check the effect of the use of waste-products as the external carbon source on biomass production. The few available studies concerning Yobs value were carried out with commercial, pure carbon sources (Majone et al., 2001). The main goal of this research was to determine the effectiveness of nitritation–denitritation during landfill leachate treatment in the SBR at a low DO concentration in the aeration phase using glycerine as the external carbon source. Moreover, sludge production under applied operational conditions was assessed.

The volumetric exchange rate was 0.3 d1. The system was operated at room temperature (20–22 °C) for 3 months. Reactors were fed by the landfill leachate with the addition of an external carbon source in form of sodium acetate (Ac) and glycerine (Gly) (CODext). The dosage of the external carbon source should provide the CODext/TKN at the beginning of the reactor cycle ca. 4.0. The chemical composition of glycerine was as follows: glycerol 80–85%, ash (NaCl) <7%, M.O.N.G. (matter organic non glycerol) <2%, methanol <0.5%, water – balance (product specification from Biodiesel Manufacturing Plant, Poland). Two solutions, one of sodium acetate and a second of glycerine were prepared in the following way: 150 g CH3COONa or 113 g of glycerine was dissolved in 1 L of distilled water which resulting in 100 mg COD/ml each. The mixture of Ac and Gly, as a feed for SBR 2 and SBR 3, were prepared in the volumetric proportion. SBR 1 was fed only by Ac, whereas SBR 2 and SBR 3 by Ac and Gly in volumetric proportion 1:3 (v/v) and 1:1 (v/v), respectively.

2.3. Chemical analyses Daily measurements of pollutant concentration in the effluent from the reactors included: chemical oxygen demand (COD), total Kjeldahl nitrogen (TKN), ammonia nitrogen, nitrites and nitrates. The activated sludge was analyzed for total suspended solids (TSS) and volatile suspended solids (VSS). In steady-state conditions, the measurements of COD, TKN, ammonia nitrogen, nitrites and nitrates during the SBR cycle were done. The analyses were performed according to APHA (1992).

3. Results and discussion 3.1. Organics and nitrogen removal kinetic

2. Methods 2.1. Landfill leachate Leachate used in the experiment originated from a well-organized landfill in Wysieka, near Bartoszyce (Warmia and Mazury district). The landfill site has been operating since 1996. Physicochemical composition of the leachate was as follows: pH 8.12 ± 0.22; COD 732 ± 36 mg O2/L; BOD5 51 ± 6 mg O2/L; BOD5/ COD 0.07; BOD5/TKN 0.09; TKN 420 ± 28 mg TKN/L; 340 ± 14 mg N–NH4/L; 48.9 ± 6.8 mg P/L; total dissolved solids 7032 ± 592 mg/L; volatile dissolved solids 1087 ± 126 mg/L, Cr 0.081 ± 0.025 mg/L; Cd 0.132 ± 0.26 mg/L; Cu 0.07 ± 0.002 mg/L; Ni 0.03 ± 0.0013 mg/L; concentrations of Pb and Hg were below detection limit. 2.2. Process configuration and system design Experiments were carried out in three SBRs (SBR 1, SBR 2, SBR 3) with a working volume of 5 L each. Reactors were equipped with a stirrer with a variable speed control system (stirrer speed was maintained at the level 36 rpm). Dissolved oxygen was supplied using porous diffusers (fine bubble aeration was used), placed at the bottom of the reactors. Moreover, the reactors were equipped with an oxygen concentration control system assuring a DO concentration in the aeration period of 0.7 ± 0.2 mg O2/L. The reactors operated in a 24 h-cycle mode. Each cycle consisted of the following phases: filling (5 min), mixing (3 h), aeration (20 h), settling (50 min) and decantation (5 min).

In the landfill, the leachate organic compound concentration (expressed as COD) was 732 mg COD/L. However, a low BOD5/ COD ratio (0.07) indicated that organics were persistent for biological treatment. The leachate originated from a landfill that has been in use for over 15 years and, therefore, showed a good correlation with the low organic compound content as low concentrations of organics in leachate from stabilized landfills is well-documented in the literature. However, recent investigations have revealed that even the leachate from young landfills contains low concentrations of organics. That may be caused by leachate recirculation which reduces the waste stabilization time, enhances gas production, and, consequently, lowers the leachate concentration, especially in terms of COD (Chan et al., 2002). Apart from refractory organics, landfill leachate contains a high nitrogen concentration. Landfilled municipal solid waste (MSW) contains high amounts of organic nitrogen in a non-degradable form as well as readily-soluble nitrogen. As a result of anaerobic digestion of MSW putrescibles, around 50% of the nitrogen undergoes solubilization (Jokela and Rintala, 2003). Due to hydrolysis of soluble protein amino acids, dipeptides or oligopeptides are formed. Fermentation of amino acid leads to the formation of organic acids and ammonia. Leachate from older landfills is rich in ammonia nitrogen due to hydrolysis and fermentation of the nitrogenous fractions of biodegradable substrates. Ammonia concentration in leachate from different landfills may vary from tens or hundreds of mg N–NH4/L (Statom et al., 2004) to 2000– 3000 mg N–NH4/L (Timur and Özturk, 1997). In the presented study, the leachate contained mainly refractory organics and a high concentration of TKN (420 ± 28 mg/L). Therefore, in order to improve nitrogen removal, the external carbon

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source was introduced to SBRs in the form of sodium acetate and glycerine at the level of about 500 mg COD/L. Determining the CODext/TKN ratio in the SBR influent at the beginning of the experiment (approximately 4.0), the possibility of the use of organic compounds from the leachate by microorganisms was not taken into account. In the start-up period (adaptation time), it appeared that the organic compound concentration in the SBR effluent (ca. 600 mg COD/L) was lower than in the raw leachate (ca. 730 mg COD/L). This indicated that some of them (besides the external carbon source) were used by microorganisms. Finally, it was assumed that CODrem/TKN (CODrem – organics removed in SBR cycle) at the beginning of the reactor cycle equaled ca. 5.0. Sun et al. (2012) showed that initial biodegradable organics to nitrogen ratio in the leachate should be adjusted to higher than 6.0, however their studies concerning nitrate reduction (denitrification). The amount of total Kjeldahl nitrogen in the leachate was 420 mg TKN/L and ammonium constituted 80% of TKN. On the basis of ammonia concentration in the start-up period, it was stated that in SBR 1 and SBR 2 with Ac and Ac:Gly (1:3, v/v), respectively, the adaptation period of the microorganisms lasted about 8 weeks (data not shown). After this time, the ammonium concentration in SBRs effluent was below 1.5 mg N–NH4/L. The start-up period for SBR 3, fed with leachate with Ac:Gly (1:1, v/v) lasted longer – 10 weeks. During the steady state conditions in all reactors, despite limited oxygen concentration in the aeration phase, ammonia concentration reached a low level (Fig. 1) and the conversion of ammonia into nitrite was elevated to 98–99%. This means that stable partial nitrification (nitritation) was achieved. Nitritation requires a reduction of the activity of the nitrite oxidizing bacteria (NOB), without affecting the ammonia-oxidizing microorganisms (AOB). This can be achieved in several ways, e.g. dissolved oxygen (DO) limitation and pH regulation. However, pH also affects the chemical equilibria of ammonium-free ammonia (FA) and nitrite-free

(a) 10

nitrous acid (FNA), which can play a critical role in nitritation (Ahn et al., 2011; Gabarró et al., 2012). Inhibitory concentrations of FA and FNA on Nitrobacter sp. ranged from 0.1 to 1.0 mg N–NH3/L and from 0.2 to 2.8 mg N–HNO2/L, respectively. In the present study free nitrous acid amounts in SBR 1–SBR 3 were unnoticeable and couldn’t be regarded as inhibitory concentrations. However, free ammonium concentration in all SBRs reached 3.1 mg N–NH3/L that resulted from slightly alkaline reaction. Thus, one of the reasons of the low formation of nitrate in the effluent might be FA inhibition of NOB. However, because of the fact that NOB may acclimate to FA to a concentration as high as 22 mg N–NH3/L (Jianlong and Ning, 2004; Villaverde et al., 2000) it was stated that in presented study the main factor responsible for the inhibition of the second nitrification phase (oxidation nitrite-to-nitrate) was low oxygen concentration in the aeration phase (0.7 mg O2/L). Nitrates constituted averagely 1.3, 1.7 and 1.3 mg N–NO3/L, in the effluent of SBR 1, SBR 2 and SBR 3, respectively (Fig. 1). Nitritation in synthetic wastewater at a low DO level was also noted by Ruiz et al. (2003). However, other studies have indicated that nitritation, besides limited oxygen, requires high temperature conditions (Jianlong and Jing, 2005). Based on ammonium, nitrites and COD concentration profiles during the SBR cycle, a kinetic analysis was performed under steady state conditions. Ammonium removal, nitritation in the aeration phase and COD removal in the mixing phase proceeded according to zero-order kinetics, which involved linear changes of ammonium, nitrite (Fig. 2) and COD concentrations over time (Fig. 3). However, COD removal in the aeration phase was described by a first-order removal rate (Fig. 3). Fig. 2 shows the ammonia, nitrite and nitrate levels over time during the SBR cycle. In the mixing phase, regardless of the share of glycerine in Ac:Gly proportion, complete denitritation was achieved, which was revealed in a lack of oxidized form of nitrogen. At the same time, a significant decrease of COD concentration was observed (Fig. 3). Organic substance removal rate and the

(b) 40 N-NO2 [mg/L]

N-NH4 [mg/L]

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8

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6

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SBR 3 2 0 0

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Fig. 1. Changes in the concentration of ammonia (a), nitrite (b) and nitrate (c) in SBR 1, SBR 2 and SBR 3.

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(a)

(b)

100

N-NH4, N-NO2, N-NO3 [mg/L]

N-NH4, N-NO2, N-NO3 [mg/L]

100 CN-NH4= - 17.26t + 99.6

80

2

R = 0.96

60 CN-NO2= 5.78t

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R = 0.96

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CN-NH4= - 17.84t + 101.4

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R = 0.97 60 CN-NO2= 3.54t

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CN-NH4= - 17.09t + 98.9

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CN-NO2= 1.44t 2

mixing phase

R = 0.87 20 0 0

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Fig. 2. Changes in ammonia, nitrite and nitrate concentrations during SBR cycle (a) SBR 1, (b) SBR 2, (c) SBR 3.

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800

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CCOD= - 67.02 t + 604.92

CCOD= - 131.6 t + 606.14

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R = 0.98

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CCOD=464.3 exp(-0.35 t)+42

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mixing phase

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3

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Fig. 3. Changes in COD concentration in mixing and aeration phase of the SBR cycle (a) SBR 1, (b) SBR 2, (c) SBR 3.

amount of COD removed depended on the proportion of Ac:Gly. The highest removal of organics, from 630 mg COD/L to

212 mg DOC/L, was noted in SBR 1 with Ac as a carbon source. In this case, the COD removal rate equaled 132 mg COD/L h. Glycerine

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addition (Ac:Gly 1:3, v/v) in SBR 2 resulted in a 2-fold lower COD removal rate in the mixing phase. COD concentration at the end of this phase was 420 mg COD/L. The lowest COD removal and process rate was observed in the reactor fed by Ac:Gly (1:1, v/v). In the aeration phase with the limited oxygen concentration, the ammonium removal rate (rNH4) was at a stable level of 17.1–17.8 mg N–NH4/L h (ca. 5 mg N–NH4/g TSS h) (Fig. 2), and ammonium concentration as in the effluent was reached after 6 h of this phase, which means that complete ammonium removal followed within 9–10 h of the SBR cycle. Concurrent with a decrease in ammonium concentration, an increase in nitrite concentration (nitritation) took place. Nitrate concentration in the reaction time did not exceed 2 mg N–NO3/L. The highest nitritation rate (5.8 mg N–NO2/L h; 1.6 mg N–NO2/g TSS h) was obtained in SBR 1 (fed by Ac) and the lowest – 1.4 mg N–NO2/ L h (0.4 mg N–NO2/g TSS h) – in SBR 3 (Ac:Gly 1:1, v/v) (Fig. 2). The comparison of ammonium concentration in the influent with oxidized nitrogen concentration in the effluent confirmed that nitrogen removal was due to nitritation–denitritation. Nitrite reduction was observed both in the mixing phase and in the aeration phase, as indicated by a significantly higher ammonium removal rate compared to the nitritation rate. In SBR 1 and SBR 2, the average ammonium removal rate was 3-fold higher than the nitrite formation rate. However, in SBR 3, rNH4 was over 12-fold higher than rNO2. It is worth noting that denitritation, occurring mainly in the aeration phase at a limited DO concentration, was the main process responsible for nitrogen removal. The low amount of nitrites reduced in the mixing phase was caused by their low concentration at the beginning of the mixing phase. COD removal in the aeration phase, other than in the mixing phase, proceeded according to first-order kinetics (Fig. 3). The lowest value of rCOD (88 mg COD/L h) was obtained in SBR 1 and in SBR 3, being almost twice higher (164 mg COD/L h). The differences in COD removal rate resulted from the initial COD concentration in the aeration phase which, in turn, was due to variable COD removal in the mixing phase. 3.2. Activated sludge biomass production Sludge production is the result of the increment of dry mass of activated sludge due to internal substrate storage in microbial cells, new cell biosynthesis and, on the other hand, the reduction of the biomass due to storage polymer degradation, cell lysis and death. In practice, net sludge production is expressed as the biomass yield coefficient (Yobs). Yobs is the mass of bacteria formed per mass of COD removed, depending on the energy requirements. The value of the observed biomass yield coefficient Yobs corresponds to the net biomass yield coefficient and can be calculated from the following equation:

Y obs ¼

X org  ðV w =tÞ þ X e  ðV d =tÞ ðC i  C e Þ  ðV d =tÞ

where Yobs – observed biomass yield coefficient (mg VSS/mg COD), Xorg – volatile suspended solids in SBR (mg VSS/L), Vw – volume of suspended solids disposed in SBR operating cycle (L), t – time of SBR operating cycle (d), Xe – effluent volatile suspended solids concentration (mg VSS/L), Vd – volume of leachate effluent/influent in SBR operating cycle (L), Ci – concentration of COD in SBR influent (mg COD/L), Ce – concentration of COD in SBR effluent (mg COD/L). The yield of activated sludge biomass growing on various substrates is inevitably associated with sludge growth and substrate utilization kinetics, therefore Yobs is an important parameter for treatment plant operators to calculate the activated sludge wastewater treatment performance. The type and dose of organic carbon source influence the fraction, type and anabolism of the biomass

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(Obaja et al., 2005). Majone et al. (2001) investigated sludge production in denitrification with pure carbon sources such as acetate, ethanol, glucose and glutamic acid. The highest value of the biomass yield coefficient (0.74 g COD/g COD) was observed when glucose was an electron donor and the lowest (Yobs – 0.56 g COD/ g COD) was for glutamic acid. Medium values were obtained for ethanol and acetate (0.7 g COD/g COD and 0.65 g COD/g COD, respectively). The presented research showed that Yobs largely depended on the contribution of the waste-products in the total external carbon source. The highest Yobs – 0.6 mg VSS/mg COD was achieved in SBR 1, fed by Ac. A slightly lower value of Yobs (0.55 mg VSS/mg COD) was obtained in SBR 2 with Ac:Gly (1:3, v/v). In SBR 3, where the share of glycerine was 50% of the total external carbon source, the biomass yield coefficient was nearly 30% lower (0.47 mg VSS/ mg COD) in comparison to SBR 1. This means that glycerine – waste product influenced reduction of biomass production. This is with accordance with the results obtained by Pitter and Chudoba (1990) that lower biomass production is observed with slowly-biodegradable organic substances as a carbon source. Contrary results were achieved by Frison et al. (2013). They investigated external carbon sources to enhance short-cut nitrification–denitrification and denitrifying phosphorus removal using SBR to treat anaerobic supernatant. According to these authors, waste-originated carbon sources, such as municipal solid waste or cattle manure with silage maize fermentation liquids lead to higher biomass yield (0.45 and 0.31 mg VSS/mg COD, respectively) than pure carbon sources (0.18 and 0.24 mg VSS/mg COD, respectively for acetic acid and glycerol). However, the value of Yobs obtained by the authors from waste-originating carbon sources are close to the results for Ac:Gly (1:1, v/v) (Yobs 0.47 mg VSS/mg COD). The data in the literature show that biomass production depends on the ratio of the initial substrate concentration to the initial biomass concentration (C0/X0). Liu (1996) analyzed the correlation between substrate consumption and biomass growth in aerobic batch tests. It was assumed that the overall consumption of substrate is the sum of the substrate consumed for growth, maintenance and energy loss. In aerobic cultures a decrease in the observed biomass coefficient by increasing the C0/X0 ratio was observed. According to the authors, it was due to energy loss under substrate-sufficient conditions. Similar results, (lowering biomass production C0/X0 increases) but under anaerobic conditions, were obtained by Moreno et al. (1999). Other authors (Chudoba et al., 1991; after Moreno et al., 1999), suggest this behavior was due to higher C0/X0 ratios having enough energy for the synthesis reactions to occur during the cellular replication cycle. In this study, initial C0/X0 at the beginning of the SBR cycle was similar in all reactors, although a variable COD removal rate in the mixing phase affected organic concentration at the beginning of the aeration phase (Fig. 3). Therefore, C0/X0 varied at this moment. The highest value of C0/X0 (0.15) was obtained in SBR 3 with Ac:Gly (1:1, v/v) and the lowest (0.063) was in SBR 1 with Ac. As with the batch tests of Liu (1996) and Moreno et al. (1999), a similar correlation between observed biomass coefficient (Yobs) and C0/X0 was noted in the presented experiment. In contrast to the studies of the above mentioned authors, carried out under substrate-sufficient batch cultivation of microorganisms, it is difficult to deduce if in the presented experiment Yobs reduction was related to (i) energy loss, metabolic uncoupling and modification of the respiratory chain and/or (ii) COD use for denitrification. 3.3. Nitrogen removal efficiency In systems with activated sludge, total nitrogen removal consists of denitrification/denitritation, preceded by nitrification/nitritation and the use of nitrogen for biomass synthesis. In the present

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Table 1 The effectiveness of nitritation, denitrification and nitrogen removal in SBR 1–SBR 3. Effectiveness [%]

SBR 1

SBR 2

SBR 3

Nitritation Denitritation Nitrogen removal

99.0 60.5 65.1

98.0 65.8 69.7

98.0 75.6 78.0

study, the lowest value of the effectiveness of nitrogen removal (65.1%) was observed in SBR 1 with Ac, the highest (78%) was in SBR 3 with Ac:Gly (1:1, v/v). Nitrogen used for biomass production (biomass synthesis) comprised of 13.5–17% of the total removed nitrogen, the rest was due to denitritation. An increase in denitritation effectiveness from 60.5% to 75.6% (Table 1) and simultaneous decrease of Yobs from 0.6 to 0.46 mg VSS/mg COD in SBR 1 with Ac and SBR 3 with Ac:Gly (1:1, v/v), respectively, may suggest that organics were used for nitrites reduction, as denitritation requires organics as electron donors. It may results from the presence of organics characterized by different biodegradability (Ac, Gly) in SBR influents. When acetate is a carbon source an intracellular storage may occur, mainly in the form of poly-b-hydroxybutyrate (PHB) (Third et al., 2003; Beccari et al., 2002). Thus, the highest COD removal rate in the mixing phase in SBR 1 may result from both nitrite reduction and storage materials accumulation. In SBR 2 and SBR 3, glycerine included in the mixture of COD, could be transformed into the easily biodegradable carbon compounds (such as N-butanol, butyrate, actetate, ethanol, 2,3-butanediol, propionate) (Viana et al., 2012) before it is involved in metabolic processes of the microorganisms. It means that acetate is only one of the many possible intermediates in glycerine transformation. Therefore, in SBR 2 and SBR 3 PHB accumulation proceeded less. However, the intermediates of the glycerine transformation may be the carbon source for denitritation, so higher effectiveness of the process was observed, with the increasing share of glycerine in the mixture of COD. An increase in nitrogen removal effectiveness from 31% to 61% after a decrease of DO concentration from 3 to 1 mg O2/L was observed by Third et al. (2003) in simultaneous nitrification and denitrification. As well as the oxygen condition, nitrogen removal due to denitrification depended on the COD/N ratio. Tora et al., 2011 indicated that the COD/N ratio needed for complete nitrite reduction depended on the type of organic carbon source (ethanol, glycerol, fermented primary sludge, landfill leachate) and ranged from 3.0 for ethanol to 8.8 for landfill leachate. In this experiment, a COD/N of ca. 5 was insufficient to obtain complete denitritation. This meant that a higher amount of organics needed to be supplied. However, in the case of glycerine, a higher COD demand is not a problem because glycerine is a waste-product that has to be managed. Presented study indicated some advantages of using glycerine – waste-product as a carbon source for denitritation (high nitrogen removal effectiveness, low biomass production). Thus, the possibility of using glycerine as a sole carbon source in nitrogen removal should be tested.

4. Conclusion Glycerine can be used as a carbon source in nitrogen removal from landfill leachate. At limited oxygen concentrations in the aeration phase, nitritation and denitritation occurred concurrently, however at COD/TKN ca. 5.0 SBR effluent contained nitrite. Further studies should focus on determination of the optimal proportion of COD/TKN or the possibility of using a higher glycerine contribution in the carbon source (or glycerine only) to obtain complete denitritation. At Ac:Gly 1:1 (v/v), biomass production diminished. The

effect of the waste products as a carbon source on sludge production is usually not considered. For this reason, more research on this point is necessary.

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