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High rate autotrophic denitrification in fluidized-bed biofilm reactors
Accepted Manuscript High rate autotrophic denitrification in fluidized-bed biofilm reactors G. Zou, S. Papirio, A.-M. Lakaniemi, S.H. Ahoranta, J.A. Puhakka PII: DOI: Reference:
S1385-8947(15)01345-5 http://dx.doi.org/10.1016/j.cej.2015.09.074 CEJ 14230
To appear in:
Chemical Engineering Journal
Received Date: Revised Date: Accepted Date:
9 July 2015 7 September 2015 12 September 2015
Please cite this article as: G. Zou, S. Papirio, A.-M. Lakaniemi, S.H. Ahoranta, J.A. Puhakka, High rate autotrophic denitrification in fluidized-bed biofilm reactors, Chemical Engineering Journal (2015), doi: http://dx.doi.org/ 10.1016/j.cej.2015.09.074
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High rate autotrophic denitrification in fluidized-bed biofilm reactors G. Zoua,*, S. Papirioa, A-M.Lakaniemia, S.H. Ahorantaa, J.A. Puhakkaa 7/9/2015 (a)
Department of Chemistry and Bioengineering, Tampere University of Technology, P.O. Box
541, FIN-33101 Tampere, Finland.
Resubmitted to: Chemical Engineering Journal (*)
Corresponding author: E-mail:
[email protected]
Telephone:
+358 40 1981267
Fax:
+358 33 641392
Abbreviations DGGE - Denaturing gradient gel electrophoresis DO - Dissolved oxygen FBRs- Fluidized-bed reactors HRT - Hydraulic retention time PBRs -Packed-bed reactors PCR - Polymerase chain reaction VS - Volatile solids
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Abstract High rate, high efficiency thiosulfate-driven autotrophic denitrification and denitritation with Thiobacillus denitrificans dominated biofilms were achieved in fluidized-bed reactors (FBRs) operated at 20.0±2.0 and 30.0±0.2°C. Complete nitrate removal was obtained even at nitrate loading rate and hydraulic retention time (HRT) of 600 mg L-1 h-1 and 10 min, respectively. Further decrease of HRT to 5 min resulted in 50% of nitrate removal efficiency. Nitrite did not accumulate when nitrate was used as electron acceptor unless HRT was decreased to 5 min. Effluent pH remained at 5.8 during denitrification. When nitrite was supplemented as the electron acceptor, denitritation effectively proceeded with the highest nitrite loading rate of 228 mg·L-1·h-1. Similar denitrification and denitritation performances were obtained at 20.0±2.0 and 30.0±0.2°C. Batch assays conducted at temperature range from 1 to 46°C, however, showed a significant impact of temperature on autotrophic denitrification. Ratkowsky model was used to estimate the minimum, optimal and maximum growth temperatures of Thiobacillus denitrificans dominated culture that were below 1, 26.6 and 50.8°C, respectively. Keywords Autotrophic denitrification; fluidized-bed biofilms; hydraulic retention time; Thiobacillus denitrificans; temperature; thiosulfate
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1. Introduction Nitrate contamination in ground and surface waters is a result of excessive use of fertilizers and disposal of improperly treated wastewaters [1, 2]. Exposure to nitrate and nitrite leads to eutrophication of aquatic environments and potential human health hazards such as methemoglobinemia and carcinogenic diseases [3, 4]. Sulfur-based autotrophic denitrification is a promising alternative to heterotrophic denitrification due to two main advantages: 1) no need of extra supplementation of organic carbon and 2) lower production of sludge [5-7]. The use of reduced sulfur compounds (i.e. S2-, So, and S2O32-) as electron donors for chemolithotrophic denitrification is particularly promising for organic-deficient waters where sulfur and nitrogen occur simultaneously [8]. However, the main disadvantages of sulfur-based autotrophic denitrification are sulfate and acidity generation [5, 6, 9]. Equations 1 and 2 demonstrate the stoichiometry of the two-step autotrophic denitrification by using thiosulfate as electron donor [10]. Equation 3 indicates the overall reaction of complete autotrophic denitrification [10]. The biosynthesis of new cells is expressed by the term C5H7O2N among the products. 2−
−
3 . 10 NO 3 + S 2 O 3
+ 0 . 73 H 2 O + 0 . 45 HCO −
→ 0 . 09 C 5 H 7 O 2 N + 3 . 1 NO 2 + 1 . 64 H
−
2 . 07 NO 2 + S 2 O 3
2−
+ 0 . 48 H
+
+
− 3
+ 0 . 09 NH
+ 2 SO 4
+ 0 . 45 HCO
− 3
−
1 . 24 NO 3 + S 2 O 3
2−
+ 0 . 11 H 2 O + 0 . 45 HCO
→ 0 . 09 C 5 H 7 O 2 N + 0 . 62 N 2 + 0 . 4 H
+
− 3
+ 2 SO 4
2−
+ 0 . 08 NH
→ 0 . 09 C 5 H 7 O 2 N + 1 . 03 N 2 + 0 . 31 H 2 O + 2 SO 4
(1)
+ 4
(2)
2−
+ 0 . 09 NH 2−
+ 4
+ 4
(3)
3
Nitrite is formed as an intermediate product of nitrate reduction and can exhibit inhibitory effects on denitrification at high concentrations [10, 11]. Autotrophic denitrification with reduced sulfur compounds (e.g. thiosulfate) is effectively conducted by Thiobacillus denitrificans and Thiomicrospira denitrificans as reported by Taylor and Hoare [12] and Hoor [13], respectively. Unlike heterotrophic denitrifiers, the activity of these bacteria is significantly affected by the operating temperature. The optimal growth temperature of Thiobacillus denitrificans is approximately 30°C [14]. A significant reduction (50-70%) of denitrification efficiency was observed at temperatures ranging between 5 and 10°C [15, 16]. However, only a few studies have reported the effects of temperature on the activity of Thiobacillus denitrificans [11, 14, 17]. In particular, the minimum and maximum growth temperatures have never been reported. Sulfur-based autotrophic denitrification for treatment of waste- and groundwater has been widely studied in batch assays and continuous packed-bed reactors (PBRs) [7, 15, 18]. Less attention has been given to fluidized-bed reactors (FBRs) that were previously demonstrated to effectively maintain heterotrophic denitrification [19, 20]. Moreover, only few studies reported the performance of autotrophic denitrification at feed nitrate below 100 mg L-1 which is comparable with the nitrate concentrations in actual organicdeficient wastewaters [16, 18, 21, 22]. The aim of the present study was to investigate the potential of fluidized-bed biofilms for performing autotrophic denitrification and denitritation with thiosulfate in terms of nitrate and nitrite removal at two different temperatures: 20±2 and 30±0.2°C. The effects of feed nitrate and nitrite concentrations and hydraulic retention times (HRTs) were evaluated in continuous experiments. Polymerase chain reaction - denaturing 4
gradient gel electrophoresis (PCR-DGGE) was used to reveal the evolution of autotrophic denitrifying microbial communities in FBRs. Finally, the effect of cultivation temperatures (1-46°C) on nitrate removal rate was studied in batch assays and modeled with Ratkowsky equation [23].
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2. Materials and methods 2.1 Microbial culture enrichment A pure culture of Thiobacillus denitrificans (DSM12475), from Leibniz-institut DSMZ (Germany), was preliminarily cultivated in batch assays and subsequently used in FBRs. The mineral medium was composed of four solutions as reported in Table 1. The trace elements provided with solution A contained FeCl3·6H2O (2.75 mg L-1), CuSO4·5H2O (0.13 mg L-1), H3BO3 (0.50 mg L-1), MnSO4·4H2O (0.64 mg L-1), Na2MoO4·2H2O (0.20 mg L-1), CoCl2·6H2O (0.15 mg L-1), ZnSO4·7H2O (0.23 mg L-1), Na2SeO4 (0.23 mg L1
). Solutions A, B and D were separately purged with N2 for 15 min and then autoclaved
at 121°C for 20 min. Solution C containing HCO3- was sterilized by filtration in a laminar hood in order to prevent CO2 formation in the autoclave. The bottles used for the batch assays were filled with the mineral medium and the pure culture of Thiobacillus denitrificans (10% v/v). Subsequently, the bottles were incubated at 27°C and placed in a gyratory shaker with a shaking speed of 200 rpm. The Thiobacillus denitrificans culture was sub-cultured 4 times prior to being used as seed for the FBRs.
2.2 Start-up and operation of FBRs Two laboratory-scale glass FBRs (volume 580 mL) were used to investigate autotrophic denitrification and denitritation by using thiosulfate as electron donor. The configuration of the FBRs was as reported by Papirio et al. [19]. FBR1 was operated at room temperature (20.0±2.0°C), whereas FBR2 was maintained at 30.0±0.2°C by means of heating wires, a temperature probe and a temperature control system. A quarter of the reactor volume was filled with granular activated carbon (particle size =
6
0.5-1 mm) used as biomass carrier in both FBRs. The FBR recirculation was maintained by a peristaltic pump and adjusted to 25% of carrier fluidization by using a flow rate of 800 mL min-1. Excess thiosulfate compared to the theoretical S2O32- consumption was used to supply sufficient electron donor for complete denitrification and denitritation (Table 2). Initially, both FBRs were filled up with N2-purged mineral medium (Table 3) and activated carbon. The medium was not sterilized and the feed was prepared by using tap water. NaHCO3 was added at a concentration of 1 g L-1and 58 mL of Thiobacillus denitrificans enrichment solution (10% of the entire FBR volume) was injected to both FBR1 and FBR2. The mineral nutrients were added at a 10-fold lower concentration than that used for growing Thiobacillus denitrificans, in order to study the biological nitrate removal at more realistic mineral nutrient levels. During the first experimental period, the FBRs were operated in fed-batch mode for 57 days in order to allow the bacterial colonization of the carrier material. Half of the FBR volume was replaced with fresh medium twice per week and the pH was continuously adjusted to 7.0. From day 58 on, the FBRs were operated in continuous mode. Between day 58 and 182, the FBRs were operated in order to test the effects of HRT, temperature and feed nitrate concentration on denitrification (Table 2, periods I and II). During period I, nitrate and thiosulfate concentrations in the feed were constantly maintained at 1230 and 2260 mg L-1, respectively. HRT was gradually decreased from 24 to 5.4 h by increasing the feed flow rate. In period II (sub-phases 1-3), feed nitrate concentration was gradually decreased from 1230 to 100 mg L-1. In order to maintain the nitrate loading rate constant at 228 mg L-1·h-1, HRT was accordingly decreased down to 0.4 h. In period II (subphases 4-5), HRT was further decreased to 10 and 5 min, keeping the feed nitrate at 100
7
mg L-1. Between period II-4 and 5, HRT was increased back to 5.4 h, in order to stabilize the denitrification process. Between day 184 and 203 (period III), nitrate supplementation was stopped and both reactors were fed with nitrite in order to assess denitritation efficiency (Table 2, period III). HRT was increased back to 5.4 h and feed nitrite was gradually increased from 100 to 1230 mg L-1. Throughout the experimentation, feed thiosulfate was changed according to the feed nitrate or nitrite concentrations, keeping the S2O32-/NO3-and S2O32/NO2- ratios constant. Effluent samples were taken averagely three times a week for nitrate, nitrite, sulfate and thiosulfate analyses.
2.3 Temperature-gradient incubations The effect of different cultivation temperatures on the activity of the FBR cultures was evaluated in a temperature gradient incubator (Terratec®) that was heated at one end and cooled at the other to create a stable temperature gradient. The tested cultivation temperatures ranged from 1 to 46°C with approximately 1.5°C intervals. The incubator was running 2 days prior to the batch assays, in order to stabilize the set temperatures. The temperature in each tube was monitored before and after experimentation by using a thermometer. The 25 mL cultivation tubes were filled with 18 mL of mineral medium (Table 3) prior to purging with N2 for 15 min and sealing with rubber septa. Nitrate and thiosulfate concentrations were 1230 and 2260 mg L-1, respectively. To ensure the desired temperature to the whole cultivation volume, the tubes were incubated in the temperature gradient incubator for 1 h prior to inoculation. After that, 2 mL of stock NaHCO3 solution was added to each tube in order to achieve a concentration of 1 g L-1 and the tubes were inoculated with 2 mL of FBR enrichment cultures (10% v/v). 8
Finally, the tubes were sealed with rubber septa and aluminium caps and incubated in the temperature gradient incubator with a speed of 150 rpm. A culture broth of 0.8 mL was taken every hour during the initial 12 hours from each tube for nitrate, nitrite, sulfate and thiosulfate analyses. The final samples were taken from each tube at 24 h. All the batch assays were carried out in duplicate. The effect of the operating temperatures on the FBR culture activity was modeled by Ratkowsky equation (Equation 4) [23] (Ratkowsky et al., 1983):
= − 1 −
(4)
where g is the generation time, T is the operating temperature (°C) and Tmin and Tmax are the theoretical minimum and maximum temperatures, respectively. The optimal temperature (Topt) was determined as the temperature that resulted in the highest value of 1/. The generation time was calculated as reported in equation 5:
=
(5)
where is the specific growth rate (h-1). The specific growth rate ( ) is usually determined as the slope of the linear part of the plot displaying the natural log of the optical density versus time. In this study, was derived from the nitrate removal rate by determining the slope of the linear part of the plot displaying the evolution of nitrate concentration against time. Finally, the experimental data, obtained at different temperatures from batch assays, were fitted with Ratkowsky equation by using OriginLab software. 9
2.4 Analytical methods The liquid samples from FBRs and batch assays were filtered through 0.45 µm ChromafilXtra PET-20125 membranes (Macherey-Nagel, Germany). Nitrate, nitrite, sulfate and thiosulfate were measured by ion chromatography (IC) as reported by Zou et al. [24]. Effluent pH and dissolved oxygen (DO) were measured directly in the FBRs as reported by Papirio et al. [25]. The volatile solids (VS) concentrations of the biofilmcoated activated carbon were measured at the initial fed-batch operation and during period II on days 151, 167 and 184, as reported by Papirio et al. [19]. The evolution of autotrophic denitrifying microbial communities during FBR operation was carried out by using PCR-DGGE technique. Samples of biofilm-coated activated carbon were taken five times during the experimentation (on days 86, 148, 168, 184 and 204). The biomass samples were sonicated and then the liquid separated from the activated carbon was filtrated with Cyclopore track etched 0.2 µm membranes (Whatman, USA), in order to detach the bacteria from activated carbon. The PCRDGGE analyses were performed as reported by Papirio et al. [19]. The dominant DGGE bands were excised from the gel, eluted in 20 µL of sterile water at 4°C overnight, and amplified by PCR for sequencing. The sequencing was performed by MacroGen (Seoul, Korea) and the obtained data was compared with National Center for Biotechnology Information database to identify the microorganisms.
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3. Results
3.1 FBR performance 3.1.1 Denitrification Figs. 1 and 2 show the profiles of HRT, nitrate, nitrite, sulfate and pH in both FBR1 (20.0±2.0°C) and FBR2 (30.0±0.2°C) during the over 200 days experimentation. During the initial fed-batch operation, Thiobacillus denitrificans effectively colonized the carrier, resulting in an immobilized VS concentration of 0.2 and 0.17 g mL-1 of activated carbon on day 57 in FBR1 and FBR2, respectively (data not shown). Autotrophic denitrification required 44 days before achieving complete nitrate removal in FBR2 (Fig. 1C). Nitrate removal reached 79% in FBR1 during this experimental phase (Fig. 1C). Thiosulfate oxidation was particularly unstable until day 54 and after which S2O32- remained below the detection limit in both FBRs (Fig. S1B). Effluent pH dropped to 5.7 and 6.7 and sulfate sharply increased to approximately 3700 and 3200 mg L-1 on days 38 and 33 in FBR1 and FBR2, respectively (Fig. 1C and D). During period I, the feed nitrate was 1230 mg/L and the HRT was gradually decreased from 24 to 5.4 h in both FBRs (Fig. 1A). Effluent nitrate and nitrite remained below the detection limit in both FBR1 and FBR2 (Fig. 1B) with a nitrate loading rate of 228 mg·L-1·h-1 (Table 2). Effluent sulfate concentrations remained stable at approximately 3500 mg L-1 in both FBR1 and FBR2 (Fig. 1C), being about 15% higher than the theoretical value of 3065 mg·L-1 which was calculated by the complete feed nitrate reduction to dinitrogen gas based on Equation 3. Thiosulfate was not detected and pH remained at 5.8 in both FBRs (Figs. S1B and 1D). Effluent DO was 0.35 and 0.27 mg L1
on average in FBR1 and FBR2, respectively (Fig. S1A).
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In period II (sub-phases 1-3), autotrophic denitrification was efficiently performed at 100 mg·L-1 of feed nitrate in both FBR1 and FBR2 at HRT gradually decreasing from 2.6 to 0.4 h, resulting in complete nitrate and nitrite removal and thiosulfate oxidation (Figs. 1B, C and 2B, C). From day 173 to day 182 (period II, sub-phases 4-5), HRT was further decreased in both FBRs first to 10 min (period II-4) and then to 5 min (period II5) obtaining a nitrate loading rate of 600 and 1200 mg·L-1·h-1, respectively, by keeping the feed nitrate at 100 mg·L-1 (Fig. 2A). In period II-4 (days 173-177), nitrate was completely removed except for a peak of 15 mg·L-1 on day 174 in FBR1. No nitrite was observed (Fig. 2B) and sulfate concentrations remained at approximately 275 mg·L-1 (Fig. 2C). In period II-5 (days 181-182), denitrification efficiency was reduced after HRT decrease to 5 min. Effluent nitrate concentrations increased up to 60 and 20 mg·L1
on day 181 and decreased to 27 and 13 mg·L-1 on day 182 in FBR1 and FBR2,
respectively (Fig. 2B), resulting in the nitrate removal efficiency of 60 and 80% on average in FBR1 and FBR2, respectively. Nitrite slightly increased to 4.6 and 2.6 mg·L1
in FBR1, whereas no nitrite was observed in FBR2. Effluent sulfate concentrations
remained at approximately 190 mg·L-1 and 265 mg·L-1 in FBR1 and FBR2, respectively. During this phase, effluent DO continuously increased up to 0.58 mg·L-1 in both FBRs (Fig. 2D). Effluent pH gradually increased from 5.8 (period I) up to approximately 7.0 during period II (Fig. 1D) and thiosulfate remained below the detection limit in both FBRs (Fig. S1B). During period II (days 148-184), VS concentration remained stable at 0.19 and 0.18 g mL-1 of biofilm-coated activated carbon in FBR1 and FBR2, respectively.
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3.1.2 Denitritation During period III, nitrite was completely removed in both FBRs at feed nitrite 100, 600 and 1230 mg·L-1 at HRT 5.4 h (Fig. 1B). Effluent sulfate concentrations remained at approximately 350, 2000 and 2800 mg·L-1 in period III-1, III-2 and III-3, respectively. This was 75, 65 and 15% higher than the corresponding stoichiometric values (Equation 2), respectively. Thiosulfate was not detected during periods III-1 and III-2, whereas S2O32- concentration increased up to 600 mg·L-1 in period III-3 (Fig. S1B). Effluent pH increased to approximately 7.2 (Fig. 1D) and DO decreased again to 0.35 and 0.27 mg·L-1 in FBR1 and FBR2, respectively (Fig. S1A).
3.2 Microbial community in the FBRs Fig. 3 shows the microbial community profiles of both FBR1 and FBR2 during the experimentation. The numbered bands on the DGGE gel indicate the excised bands that were successfully sequenced. Table 4 reports the evolution of autotrophic denitrifying microbial cultures in both reactors. Results indicate that almost 90% of the sequenced bands represented Thiobacillus denitrificans (Fig. 3 and Table 4). In FBR1 operated at 20.0±2.0°C, Thiobacillus denitrificans and Chryseobacterium hispalense were detected on day 86 (bands 1 and 2, respectively). From day 148 on, only Thiobacillus denitrificans was observed (Table 4 and Fig. 3). In FBR2 operated at 30.0±0.2°C, more microbial species were detected (Table 4) whilst Thiobacillus denitrificans was the dominant species throughout FBR operation. On day 86, besides Thiobacillus denitrificans (band 1), Sediminibacterium salmoneum (bands 3, 4, 5 and 6), Chryseobacterium hispalense (band 7) and Dyella thiooxydans (bands 8 and 9) were
13
observed (Fig. 3 and Table 4). On days 148 and 168, Dyella thiooxydans (bands 10 and 15) was still present, whereas it remained below the DGGE detection on days 184 and 204. In addition, Sediminibacterium salmoneum and Chryseobacterium hispalense faded away from day 148 onwards.
3.3 Modeling of the temperature response by Ratkowsky equation Fig. 4 shows an example of nitrate and nitrite profiles in batch assays at three temperatures (6, 23 and 46°C) that were selected as representatives for all the tested temperatures. At 23°C, nitrate was completely removed in 12 h. However, nitrate consumption was significantly lower at 6 and 46°C, resulting in 25 and 17% of nitrate removal in 12 h and 46 and 26% of nitrate removal in 24 h, respectively. Nitrite accumulation was observed at all temperatures. NO2- increased to approximately 500 mg·L-1 after 12 h at 23°C prior to being completely removed by 24 h. At 6 and 46°C, nitrite accumulated up to 355 and 110 mg·L-1 by 24 h, respectively. Both nitrate and nitrite evolution indicated that complete N removal was not achieved at 6 and 46°C. The experimental data successfully fitted with Ratkowsky equation. Fig. 5 shows the modeling curve and the fitted parameters of b, c, Tmin, Tmax and Topt. The optimal temperature was 26.6°C, in correspondence of the peak of the modeling curve. The minimum (Tmin) and maximum (Tmax) temperatures were estimated as below 1 and 50.8°C, respectively, and determined as the temperature values where √1/ = 0.
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4. Discussion
This study revealed that autotrophic denitrification in terms of nitrate and nitrite removal can be effectively maintained in two FBRs by using granular activated carbon and thiosulfate as biomass carrier and electron donor, respectively, and a pure culture of Thiobacillus denitrificans as a seed. At the highest feed nitrate of 1230 mg·L-1, nitrate loading rate reached 228 mg·L-1 h-1 (51.5 N-NO3- mg L-1 h-1) which is comparable with the previous studies (Table 5). Sulfate was constantly produced by thiosulfate oxidation and SO42- concentrations were higher than the theoretical ones. This discrepancy was likely due to the observation of other thiosulfate oxidizers (i.e. Dyella thiooxydans) which can utilize dissolved oxygen as an electron acceptor [32], resulting in the oxidation of the excess feed thiosulfate. Thiosulfate-driven autotrophic denitrification produced acidity resulting in a decrease of pH to approximately 5.8 in both FBRs. The external alkalinity supplemented as NaHCO3 (1 g L-1) was not sufficient to maintain neutral conditions in the reactors. In order to reduce 1230 mg·L-1 of nitrate, the stoichiometric amount of NaHCO3 consumed as both carbon source and alkalinity is 1.14 g·L-1and 1.67 g·L-1 according to Mora et al. [10] (Equation 3) and Chung et al. [6], respectively. Also, the oxidation of excess thiosulfate compared to the theoretical S2O32- consumption in the FBRs by other thiosulfate oxidizers (i.e. Dyella thiooxydans) generated acidity and might have consumed part of the supplemented alkalinity. Nevertheless, microbial cultures were effectively adapted to the slightly acidic conditions within the FBRs. To the best of our knowledge, this is the first study reporting continuous-flow autotrophic denitrification at pH 5.8, in contrast with Moon et al. [33] and Koenig and Liu [34] who reported that autotrophic denitrification with elemental sulfur was completely inhibited at pH 6. 15
After operation at high nitrate concentrations in order to achieve high biomass colonization on the carrier, FBRs were operated at feed nitrate of 100 mg·L-1 to evaluate the process robustness under more realistic conditions for nitrate contaminated wastewaters. Efficient denitrification was maintained even at HRT of 10 min, which has never been reported previously (Table 5). The nitrate loading rate reached the highest of 600 mg·L-1 h-1 (Table 5, 135.4 N-NO3- mg L-1 h-1). The detection of residual nitrate and nitrite in the effluent at a HRT of 5 min indicated that FBRs approached the maximum loading capacity, as Sierra-Alvarez et al. [18] reported that nitrate or nitrite accumulation is a reliable marker of reactor overloading. This high denitrification efficiency was due to the effective mass transfer of the substrates to microorganisms of the FBRs [21]. At feed nitrate of 100 mg L-1, sulfate production was still higher than the stoichiometric value due to the excess of thiosulfate in the feed and the higher DO levels caused by the increase of the feed flow rate. Effluent pH increased to approximately 7.0 due to decreased acidity production during this phase, likely providing favorable conditions for autotrophic denitrification at such short HRTs [34]. The FBR biofilm cultures also demonstrated to efficiently remove nitrite when it was used as the sole electron acceptor, as previously reported by Sun and Nemati [7] and Zhou et al. [16]. In the batch assays, nitrite accumulated as an intermediate of denitrification at all temperatures. This was due to the delay in the activation of the nitrite-reducing enzymes or the lower specific nitrite utilization rate [10]. When nitrate was fed to the reactors, nitrite was below the detection limit except when FBRs were operated at the maximum nitrate loading. FBRs permitted an efficient contact between the microorganisms and substrate that allowed a faster removal of nitrite.
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During period III, nitrite was fed to both reactors, resulting in complete nitrite removal at HRT 5.4 h. Nitrite loading rate reached the highest of 228 mg L-1 h-1 which was comparable with nitrate loading rate during denitrification. Autotrophic denitritation consumed acidity (Equation 2) that resulted in the increase of effluent pH to 7.2, in agreement with Chung et al. [6]. The S2O32-/NO2- ratio used was much higher than the theoretical ratio of 0.5 mol/mol (Equation 2) and resulted in 600 mg L-1 of effluent thiosulfate at feed nitrite of 1230 mg L-1. Temperature has been shown to significantly affect the performance of autotrophic denitrification in pilot and full scale applications [15, 16]. In the present study, FBR denitrification with thiosulfate was efficiently maintained at both 20±2 and 30±0.2°C. Some differences in the behaviors of the two reactors were observed during the initial fed-batch operation and at the lowest HRTs used (10 and 5 min), demonstrating that the process was more favorable at 30°C. The optimal temperature of nitrate removal obtained by Ratkowsky modeling (26.6°C) also confirmed that denitrification efficiency was the highest at temperatures close to 28-32°C as reported by Kelly and Wood [14]. Furthermore, the fact that the optimal temperature (26.6°C) was close to the operating temperatures of both FBRs accounts for the comparable denitrification performances of FBR1 and FBR2. The minimum and maximum growth temperatures (Tmin = below 1°C and Tmax = 50.8°C) for Thiobacillus denitrificans dominated culture were also obtained in this study and have not been previously reported elsewhere. In contrast, Oh et al. [17] demonstrated that denitrification was completely inhibited at 40°C by using a mixed autotrophic denitrifying culture. To our knowledge, the temperature dependency of nitrate removal was for the first time thoroughly determined in this study for an autotrophic denitrifying culture. Ratkowsky
17
equation has been previously applied to model the effect of temperature on iron and sulfur oxidizing microorganisms [35]. In that study, the specific growth rate (µ) (Equation 5) was derived from Fe2+ oxidation or SO42- production rates due to the largely proportional relationship between bacteria growth rate and substrate utilization or product formation rates. Similarly, the growth rate of autotrophic denitrifying organisms has been reported to be largely proportional to nitrate utilization rate [17, 36]. Therefore, µ in Ratkowsky equation was considered as the nitrate removal rate. Nevertheless, autotrophic denitrification contains two steps in which nitrate is first reduced to nitrite and then to dinitrogen gas. This study demonstrated the effect of temperature range on nitrate removal rate of autotrophic denitrification by Ratkowsky model whilst the effect of temperature range on nitrite removal rate will require further study. The evolution of microbial communities in the FBRs revealed the dominance of Thiobacillus denitrificans even after 204 days operation with non-sterilized growth medium. This species demonstrated to be able to reduce both nitrate and nitrite under chemolithotrophic conditions with thiosulfate as electron donor. The initial presence of Sediminibacterium salmoneum [37], Chryseobacterium hispalense [38] and Dyella thiooxydans [32] was due to bacterial contamination of the open FBR systems. In contrast with Moon et al. [33], sulfate reducing bacteria were not detected despite the presence of high sulfate concentrations and the possible organic debris from bacterial lyses.
18
5. Conclusions High-rate, high-efficiency autotrophic denitrification was achieved in fluidized-bed biofilms. The following specific conclusions can be drawn for this study: 1) Autotrophic denitrification and denitritation with thiosulfate as electron donor were effectively maintained in two FBRs operated at 20.0±2.0 and 30.0±0.2°C. The highest nitrate and nitrite loading rates were 600 and 228 mg L-1·h-1, respectively. 2) Autotrophic denitrification was successfully operated even at HRT 10 min, resulting in complete nitrate and nitrite removal. 3) The microbial communities of both FBRs inoculated with a pure culture of Thiobacillus denitrificans were dominated by T. denitrificans even after 204 days operation with non-sterilized growth medium and tolerated pHs as low as 5.8. 4) Ratkowsky model was successfully used for the first time to simulate the effect of temperature on the nitrate removal efficiency of Thiobacillus denitrificans dominated biofilm. The minimum, optimal and maximum temperatures according to the model were below 1, 26.6 and 50.8°C, respectively.
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6. References [1] A. Bhatnagar, E. Kumar, M. Sillanpää, Nitrate removal from water by nanoalumina: Characterization and sorption studies, Chem. Eng. J. 163 (2010) 317-323.
[2] J.Y. Park and Y.J. Yoo, Biological nitrate removal in industrial wastewater treatment: which electron donor we can choose, Appl. Microbiol. Biotechnol. 82 (2009) 415-429.
[3] C. Chow and C. Hong, Dietary vitamin E and selenium and toxicity of nitrite and nitrate, Toxicology 180 (2002) 195-207.
[4] L. Knobeloch, B. Salna, A. Hogan, J. Postle, H. Anderson, Blue babies and nitratecontaminated well water, Environ. Health Perspect. 108 (2000) 675-678.
[5] F. Di Capua, S. Papirio, P.N.L. Lens, G. Esposito, Chemolithotrophic denitrification in biofilm reactors, Chem. Eng. J. 280 (2015) 643-657.
[6] J. Chung, K. Amin, S. Kim, S. Yoon, K. Kwon, W. Bae, Autotrophic denitrification of nitrate and nitrite using thiosulfate as an electron donor, Water Res. 58 (2014) 169178.
[7] Y. Sun and M. Nemati, Evaluation of sulfur-based autotrophic denitrification and denitritation for biological removal of nitrate and nitrite from contaminated waters, Bioresour. Technol. 114 (2012) 207-216.
[8] K. Tang, S. An, M. Nemati, Evaluation of autotrophic and heterotrophic processes in biofilm reactors used for removal of sulphide, nitrate and COD, Bioresour. Technol. 101 (2010) 8109-8118.
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[9] E. Sahinkaya, N. Dursun, A. Kilic, S. Demirel, S. Uyanik, O. Cinar, Simultaneous heterotrophic and sulfur-oxidizing autotrophic denitrification process for drinking water treatment: control of sulfate production, Water Res. 45 (2011) 6661-6667.
[10] M. Mora, A. Guisasola, X. Gamisans, D. Gabriel, Examining thiosulfate-driven autotrophic denitrification through respirometry, Chemosphere 113 (2014) 1-8.
[11] C. Fajardo, M. Mora, I. Fernández, A. Mosquera-Corral, J. L. Campos, R. Méndez, Cross effect of temperature, pH and free ammonia on autotrophic denitrification process with sulphide as electron donor, Chemosphere 97 (2014) 10-15.
[12] B. F. Taylor and D. S. Hoare, Thiobacillus denitrificans as an obligate chemolithotroph, Arch. Microbiol. 80 (1971) 262-276.
[13] A. T. Hoor, A new type of thiosulphate oxidizing, nitrate reducing microorganism: Thiomicrospira denitrificans sp. Nov., Neth. J. Sea Res. 9 (1975) 344-350.
[14] D.P. Kelly and A.P. Wood, Confirmation of Thiobacillus denitrificans as a species of the genus Thiobacillus, in the beta-subclass of the Proteobacteria, with strain NCIMB 9548 as the type strain, Int. J. Syst. Evol. Microbiol. 50 (2000) 547-550.
[15] E. Sahinkaya, A. Kilic, B. Duygulu, Pilot and full scale applications of sulfur-based autotrophic denitrification process for nitrate removal from activated sludge process effluent, Water Res. 60 (2014) 210-217.
[16] W. Zhou, Y. Sun, B. Wu, Y. Zhang, M. Huang, T. Miyanaga, Z. Zhang, Autotrophic denitrification for nitrate and nitrite removal using sulfur-limestone, J. Environ. Sci. 23 (2011) 1761-1769.
21
[17] S. Oh, K. Kim, H. Choi, J. Cho, I. Kim, Kinetics and physiological characteristics of autotrophic denitrification by denitrifying sulfur bacteria, Water Sci. Technol. 42 (2000) 59-68.
[18] R. Sierra-Alvarez, R. Beristain-Cardoso, M. Salazar, J. Gómez, E. Razo-Flores, J. A. Field, Chemolithotrophic denitrification with elemental sulfur for groundwater treatment, Water Res. 41 (2007) 1253-1262.
[19] S. Papirio, A. Ylinen, G. Zou, M. Peltola, G. Esposito, J. A. Puhakka, Fluidizedbed denitrification for mine waters. Part I: low pH and temperature operation, Biodegradation 25 (2014) 425-435.
[20] G. Zou, S. Papirio,, E. Van Hullebusch, J.A. Puhakka, Fluidized-bed denitrification of mining water tolerates high nickel concentrations, Bioresour. Technol. 179 (2015) 284-290.
[21] H. Kim, I. Lee, J. Bae, Performance of a sulphur-utilizing fluidized bed reactor for post-denitrification, Process Biochem. 39 (2004) 1591-1597.
[22] M. Soares, Denitrification of groundwater with elemental sulfur, Water Res. 36 (2002) 1392-1395.
[23] D.A. Ratkowsky, R.K. Lowry, T.A. McMeekin, A.N. Stokes, R.E. Chandler, Model for bacterial culture growth rate throughout the entire biokinetic temperature range, J. Bacteriol. 154 (1983) 1222-1226.
[24] G. Zou, S. Papirio, A. Ylinen, F. Di Capua, A-M. Lakaniemi, J.A. Puhakka, Fluidized-bed denitrification for mine waters. Part II: effects of Ni and Co, Biodegradation 25 (2014) 417-423. 22
[25] S. Papirio, G. Zou, A. Ylinen, F. Di Capua, F. Pirozzi, J.A. Puhakka, Effect of arsenic on nitrification of simulated mining water, Bioresour. Technol. 164 (2014) 149154.
[26] E. Sahinkaya, A. Kilic, Heterotrophic and elemental-sulfur-based autotrophic denitrification processes for simultaneous nitrate and Cr(VI) reduction, Water Res. 50 (2014) 278-286.
[27] H.S. Moon, K. Nam, J.Y. Kim, A long-term performance test on an autotrophic denitrification column for application as a permeable reactive barrier, Chemosphere 73 (2008) 723-728. [28] J. Gu, W. Qiu, A. Koenig, Y. Fan, Removal of high NO3- concentrations in saline water through autotrophic denitrification by the bacterium Thiobacillus denitrificans strain MP, Water Sci. Technol. 49 (2004) 105-112.
[29] A. Koenig, L. Liu, Kinetic model of autotrophic denitrification in sulphur packedbed reactors, Water Res. 35 (2001) 1969-1978.
[30] S. Oh, Y. Yoo, J. Young, I. Kim, Effect of organics on sulfur-utilizing autotrophic denitrification under mixotrophic conditions, J. Biotechnol. 92 (2001) 1-8.
[31] E. Sahinkaya, N. Dursun, Use of elemental sulfur and thiosulfate as electron donor for water denitrification, Bioprocess Biosyst. Eng. 38 (2015) 531-541.
[32] R. Anandham, S-W. Kwon, P.I. Gandhi, S-J. Kim, H-Y. Weon, Y-S. Kim, T-M. Sa, Y-K. Kim, H-J. Jee, Dyella thiooxydans sp. nov., a facultatively chemolithotrophic, thiosulfate-oxidizing bacterium isolated from rhizosphere soil of sunflower (Helianthus annuus L.), Int. J. Syst. Evol. Microbiol. 61 (2011) 392-398. 23
[33] H.S. Moon, K-H. Ahn, S. Lee, K. Nam, J.Y. Kim, Use of autotrophic sulfuroxidizers to remove nitrate from bank filtrate in a permeable reactive barrier system, Environ. Pollut. 129 (2004) 499-507.
[34] A. Koenig and L. Liu, Use of limestone for pH control in autotrophic denitrification: continuous flow experiments in pilot-scale packed bed reactors, J. Biotechnol. 99 (2002) 161-171.
[35] P. Franzmann, C. Haddad, R. Hawkes, W. Robertson, J. Plumb, Effects of temperature on the rates of iron and sulfur oxidation by selected bioleaching Bacteria and Archaea: Application of the Ratkowsky equation, Miner. Eng. 18 (2005) 13041314.
[36] H. Zeng and T.C. Zhang, Evaluation of kinetic parameters of a sulfur–limestone autotrophic denitrification biofilm process, Water Res. 39 (2005) 4941-4952.
[37] J.H. Qu and H.L. Yuan, Sediminibacterium salmoneum gen. nov., sp. nov., a member of the phylum Bacteroidetes isolated from sediment of a eutrophic reservoir, Int. J. Syst. Evol. Microbiol. 58 (2008) 2191-2194.
[38] M.C. Montero-Calasanz, M. Goker, M. Rohde, C. Sproer, P. Schumann, H.J. Busse, M. Schmid, B.J. Tindall, H.P. Klenk, M. Camacho, Chryseobacterium hispalense sp. nov., a plant-growth-promoting bacterium isolated from a rainwater pond in an olive plant nursery, and emended descriptions of Chryseobacterium defluvii, Chryseobacterium indologenes, Chryseobacterium wanjuense and Chryseobacterium gregarium, Int. J. Syst. Evol. Microbiol. 63 (2013) 4386-4395.
24
Figures Figure 1- The profiles of HRT and effluent nitrate, nitrite, sulfate concentrations and pH throughout the FBR1 and FBR2 operation. The theoretical sulfate concentration was calculated based on the complete feed nitrate and nitrite reduction to dinitrogen gas according to equation 1 and 3, respectively. Figure 2- The profiles of HRT and effluent nitrate, nitrite, sulfate and DO concentrations in the period II-3, 4 and 5 of FBR1 and FBR2 operations. The theoretical sulfate concentration was calculated based on the complete feed nitrate reduction to dinitrogen gas according equation 1. Figure 3 - Bacterial community profiles in FBR1 and FBR2 on days 86, 148, 168, 184 and 204. Figure 4 - Nitrate and nitrite evolution in batch assays at three temperatures 6, 23 and 46°C. Figure 5 - The modeling curve by Ratkowsky equation showing the relationship of temperature to nitrate removal in autotrophic denitrification. Figure S1- The profiles of effluent DO and thiosulfate throughout FBR1 and FBR2 operations.
25
26
27
28
29
30
Table 1 - The composition of the medium used for activation of Thiobacillus denitrificans. Solution A
Concentration
KH2PO4
2.0 g L-1
KNO3
2.0 g L-1
NH4Cl
1.0 g L-1
MgSO4×7H2O
0.8 g L-1
Trace elements
*
pH adjusted to 7.0 with NaOH Solution B Na2S2O3×5H2O
5 g L-1
Solution C NaHCO3
1.0 g L-1
Solution D FeSO4×7H2O
2.0 mg L-1
H2SO4 (0.1 N)
1.0 mL L-1
*The concentrations of trace elements were as presented in the text (section 2.1).
31
Table 2 - Operational conditions and feed composition of FBR1 and FBR2 at different experimental periods.
2260
NO3- or NO2loading rate (mg L-1·h-1) 51
S2O32- /NO3or NO2- ratio (mol/mol) 1.0
0
2260
77
1.0
1230
0
2260
123
1.0
6.4
1230
0
2260
192
1.0
141 − 147
5.4
1230
0
2260
228
1.0
II-1
148 − 158
2.6
600
0
1105
228
1.0
II-2
159 − 165
0.9
200
0
368
228
1.0
II-3
166 − 172
0.4
100
0
184
228
1.0
II-4
173 − 177
0.17
100
0
184
600
1.0
II-5
181 − 182
0.08
100
0
184
1200
1.0
III-1
184 − 189
5.4
0
100
219
19
0.9
III-2
190 − 196
5.4
0
600
1314
111
0.9
III-3
197 − 204
5.4
0
1230
2694
228
0.9
Period
Time (days)
HRT (h)
NO3(mg L-1)
NO2(mg L-1)
S2O32(mg L-1)
I-1
58 − 71
24
1230
0
I-2
72 − 85
16
1230
I-3
86 − 123
10
I-4
124 − 140
I-5
32
Table 3 - The composition of the mineral medium used in the FBRs. Compound
Concentration (g L-1)
KH2PO4
0.2
NH4Cl
0.1
MgSO4×7H2O
0.08
Trace elements
*
FeSO4×7H2O
2 mg L-1
KNO3
2
Na2S2O3×5H2O
5
NaHCO3
1 pH was adjusted to 7.0
*The concentrations of trace elements were 10 times lower than that used for activating Thiobacillus denitrificans, as explained in section 2.1.
33
Table 4 - The identity of the sequenced DGGE bands (16SrDNA) based on the biomass samples taken from FBR1 and FBR2. FBRs
Similarity (%)
Phylogenetic group (class/order/family)
Thiobacillus denitrificans
Accession Number NR_025358.1
100
β/Hydrogenophilales/Hydrogenophilaceae
2
Chryseobacterium hispalense
KM199279.1
100
Flavobaceriia/Flavobacteriales/Flavobacteriaceae
148
1
Thiobacillus denitrificans
NR_025358.1
100
β/Hydrogenophilales/Hydrogenophilaceae
168
1,11
Thiobacillus denitrificans
NR_025358.1
99.8-100
β/Hydrogenophilales/Hydrogenophilaceae
184
16,17,18,19,20
Thiobacillus denitrificans
NR_025358.1
99.5-100
β/Hydrogenophilales/Hydrogenophilaceae
204
25, 26, 27,28
Thiobacillus denitrificans
NR_025358.1
99.8-100
β/Hydrogenophilales/Hydrogenophilaceae
1
Thiobacillus denitrificans
NR_025358.1
100
β/Hydrogenophilales/Hydrogenophilaceae
KF358456.1
99.8-100
7
Sediminibacterium salmoneum Chryseobacterium hispalense
KM199279.1
100
Sphingobacteriia/Sphingobacteriales/Chitinophagac eae Flavobaceriia/Flavobacteriales/Flavobacteriaceae
8,9
Dyella thiooxydans
NR_116006.1
99.8-100
Flavobaceriia/Flavobacteriales/Flavobacteriaceae
1
Thiobacillus denitrificans
NR_025358.1
100
β/Hydrogenophilales/Hydrogenophilaceae
10 12,13,14
Dyella thiooxydans
NR_116006.1
99.8-100
Flavobaceriia/Flavobacteriales/Flavobacteriaceae
Thiobacillus denitrificans
NR_025358.1
100
β/Hydrogenophilales/Hydrogenophilaceae
15
Dyella thiooxydans
NR_116006.1
99.6
Flavobaceriia/Flavobacteriales/Flavobacteriaceae
184
16,21,22,23,24
Thiobacillus denitrificans
NR_025358.1
99.3-100
β/Hydrogenophilales/Hydrogenophilaceae
204
29,30,31
Thiobacillus denitrificans
NR_025358.1
99.1-100
β/Hydrogenophilales/Hydrogenophilaceae
Operation day
Band label
Closest species in GenBank database
1 86 FBR1 (20.0±2.0°C)
86
FBR2 (30.0±0.2°C)
148 168
3,4,5,6
Table 5 Performance of biofilm reactors for sulfur-based autotrophic denitrification as reported in literature.
Culture
Feed N (mg NNO3-/L)
T (°C)
Electron donor
HRT (h)
N-NO3removal rate (mg N-NO3-/ L h)
28-30
S0
31.2-
2.8-4.3
[26]
13-26
0
1.3-10.0
[15]
References
Packed-bed reactor (PBR) Activated sludge
50-75
12.0 Denitrifying
30-60
S
3.0-6.0
activated sludge Mixed culture
(pilot scale) 0
144-748
23-25
S
0.4-3.0
245.0-338.8
[7]
10-100
5-32
S0
3.0-6.0
3.3-13.3
[16]
Enrichment from activated sludge
60
20
S0
24.048.0
1.3-2.5
[27]
Enrichment from
18-102
30±2
S0
1.8-30.5
10.1-12.5
[18]
250
22±2
S0
14.3-
17.1
[28]
60-251
20-25
0
S
5.5-8.9
11.3-36.3
[34]
60-400
20-25
S0
4.0-14.0
20.0-32.1
[29]
100
20-25
S0
7.1-1.5
14.1-60.0
[30]
28-30
S0
5.0
2.9-8.6
[31]
0.2-6.1
109.4-147.5
[21]
0.1-5.4
51.5-135.4
Present study
from oil reservoir Municipal activated sludge
anaerobic sludge Thiobacillus
30.5
denitrificans Thiobacillus denitrificans Thiobacillus denitrificans Activated sludge
Fluidized-bed reactor (FBR) Activated sludge Enrichment from
25-75 20-700
20
23-278
20±2;
0
S
/S2O32-
a tidal flat Thiobacillus denitrificans
S2O32-
30
35
High rate autotrophic denitrification in fluidized-bed biofilm reactors G. Zou, S. Papirio, A-M. Lakaniemi, S.H. Ahoranta, J.A. Puhakka
7/9/2015
HIGHLIGHTS -
High rate, high efficiency autotrophic denitrification was demonstrated in a FBR
-
Complete nitrate and nitrite removal was achieved at HRTs as low as 10 min
-
The nitrate and nitrite loading rates reached 600 and 228 mg·L-1·h-1, respectively
-
The biofilms were dominated by T. denitrificans and tolerated pHs as low as 5.8
-
The optimal temperature for nitrate removal was 26.6°C based on Ratkowsky modeling
36
S1385-8947(15)01345-5 http://dx.doi.org/10.1016/j.cej.2015.09.074 CEJ 14230
To appear in:
Chemical Engineering Journal
Received Date: Revised Date: Accepted Date:
9 July 2015 7 September 2015 12 September 2015
Please cite this article as: G. Zou, S. Papirio, A.-M. Lakaniemi, S.H. Ahoranta, J.A. Puhakka, High rate autotrophic denitrification in fluidized-bed biofilm reactors, Chemical Engineering Journal (2015), doi: http://dx.doi.org/ 10.1016/j.cej.2015.09.074
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
High rate autotrophic denitrification in fluidized-bed biofilm reactors G. Zoua,*, S. Papirioa, A-M.Lakaniemia, S.H. Ahorantaa, J.A. Puhakkaa 7/9/2015 (a)
Department of Chemistry and Bioengineering, Tampere University of Technology, P.O. Box
541, FIN-33101 Tampere, Finland.
Resubmitted to: Chemical Engineering Journal (*)
Corresponding author: E-mail:
[email protected]
Telephone:
+358 40 1981267
Fax:
+358 33 641392
Abbreviations DGGE - Denaturing gradient gel electrophoresis DO - Dissolved oxygen FBRs- Fluidized-bed reactors HRT - Hydraulic retention time PBRs -Packed-bed reactors PCR - Polymerase chain reaction VS - Volatile solids
1
Abstract High rate, high efficiency thiosulfate-driven autotrophic denitrification and denitritation with Thiobacillus denitrificans dominated biofilms were achieved in fluidized-bed reactors (FBRs) operated at 20.0±2.0 and 30.0±0.2°C. Complete nitrate removal was obtained even at nitrate loading rate and hydraulic retention time (HRT) of 600 mg L-1 h-1 and 10 min, respectively. Further decrease of HRT to 5 min resulted in 50% of nitrate removal efficiency. Nitrite did not accumulate when nitrate was used as electron acceptor unless HRT was decreased to 5 min. Effluent pH remained at 5.8 during denitrification. When nitrite was supplemented as the electron acceptor, denitritation effectively proceeded with the highest nitrite loading rate of 228 mg·L-1·h-1. Similar denitrification and denitritation performances were obtained at 20.0±2.0 and 30.0±0.2°C. Batch assays conducted at temperature range from 1 to 46°C, however, showed a significant impact of temperature on autotrophic denitrification. Ratkowsky model was used to estimate the minimum, optimal and maximum growth temperatures of Thiobacillus denitrificans dominated culture that were below 1, 26.6 and 50.8°C, respectively. Keywords Autotrophic denitrification; fluidized-bed biofilms; hydraulic retention time; Thiobacillus denitrificans; temperature; thiosulfate
2
1. Introduction Nitrate contamination in ground and surface waters is a result of excessive use of fertilizers and disposal of improperly treated wastewaters [1, 2]. Exposure to nitrate and nitrite leads to eutrophication of aquatic environments and potential human health hazards such as methemoglobinemia and carcinogenic diseases [3, 4]. Sulfur-based autotrophic denitrification is a promising alternative to heterotrophic denitrification due to two main advantages: 1) no need of extra supplementation of organic carbon and 2) lower production of sludge [5-7]. The use of reduced sulfur compounds (i.e. S2-, So, and S2O32-) as electron donors for chemolithotrophic denitrification is particularly promising for organic-deficient waters where sulfur and nitrogen occur simultaneously [8]. However, the main disadvantages of sulfur-based autotrophic denitrification are sulfate and acidity generation [5, 6, 9]. Equations 1 and 2 demonstrate the stoichiometry of the two-step autotrophic denitrification by using thiosulfate as electron donor [10]. Equation 3 indicates the overall reaction of complete autotrophic denitrification [10]. The biosynthesis of new cells is expressed by the term C5H7O2N among the products. 2−
−
3 . 10 NO 3 + S 2 O 3
+ 0 . 73 H 2 O + 0 . 45 HCO −
→ 0 . 09 C 5 H 7 O 2 N + 3 . 1 NO 2 + 1 . 64 H
−
2 . 07 NO 2 + S 2 O 3
2−
+ 0 . 48 H
+
+
− 3
+ 0 . 09 NH
+ 2 SO 4
+ 0 . 45 HCO
− 3
−
1 . 24 NO 3 + S 2 O 3
2−
+ 0 . 11 H 2 O + 0 . 45 HCO
→ 0 . 09 C 5 H 7 O 2 N + 0 . 62 N 2 + 0 . 4 H
+
− 3
+ 2 SO 4
2−
+ 0 . 08 NH
→ 0 . 09 C 5 H 7 O 2 N + 1 . 03 N 2 + 0 . 31 H 2 O + 2 SO 4
(1)
+ 4
(2)
2−
+ 0 . 09 NH 2−
+ 4
+ 4
(3)
3
Nitrite is formed as an intermediate product of nitrate reduction and can exhibit inhibitory effects on denitrification at high concentrations [10, 11]. Autotrophic denitrification with reduced sulfur compounds (e.g. thiosulfate) is effectively conducted by Thiobacillus denitrificans and Thiomicrospira denitrificans as reported by Taylor and Hoare [12] and Hoor [13], respectively. Unlike heterotrophic denitrifiers, the activity of these bacteria is significantly affected by the operating temperature. The optimal growth temperature of Thiobacillus denitrificans is approximately 30°C [14]. A significant reduction (50-70%) of denitrification efficiency was observed at temperatures ranging between 5 and 10°C [15, 16]. However, only a few studies have reported the effects of temperature on the activity of Thiobacillus denitrificans [11, 14, 17]. In particular, the minimum and maximum growth temperatures have never been reported. Sulfur-based autotrophic denitrification for treatment of waste- and groundwater has been widely studied in batch assays and continuous packed-bed reactors (PBRs) [7, 15, 18]. Less attention has been given to fluidized-bed reactors (FBRs) that were previously demonstrated to effectively maintain heterotrophic denitrification [19, 20]. Moreover, only few studies reported the performance of autotrophic denitrification at feed nitrate below 100 mg L-1 which is comparable with the nitrate concentrations in actual organicdeficient wastewaters [16, 18, 21, 22]. The aim of the present study was to investigate the potential of fluidized-bed biofilms for performing autotrophic denitrification and denitritation with thiosulfate in terms of nitrate and nitrite removal at two different temperatures: 20±2 and 30±0.2°C. The effects of feed nitrate and nitrite concentrations and hydraulic retention times (HRTs) were evaluated in continuous experiments. Polymerase chain reaction - denaturing 4
gradient gel electrophoresis (PCR-DGGE) was used to reveal the evolution of autotrophic denitrifying microbial communities in FBRs. Finally, the effect of cultivation temperatures (1-46°C) on nitrate removal rate was studied in batch assays and modeled with Ratkowsky equation [23].
5
2. Materials and methods 2.1 Microbial culture enrichment A pure culture of Thiobacillus denitrificans (DSM12475), from Leibniz-institut DSMZ (Germany), was preliminarily cultivated in batch assays and subsequently used in FBRs. The mineral medium was composed of four solutions as reported in Table 1. The trace elements provided with solution A contained FeCl3·6H2O (2.75 mg L-1), CuSO4·5H2O (0.13 mg L-1), H3BO3 (0.50 mg L-1), MnSO4·4H2O (0.64 mg L-1), Na2MoO4·2H2O (0.20 mg L-1), CoCl2·6H2O (0.15 mg L-1), ZnSO4·7H2O (0.23 mg L-1), Na2SeO4 (0.23 mg L1
). Solutions A, B and D were separately purged with N2 for 15 min and then autoclaved
at 121°C for 20 min. Solution C containing HCO3- was sterilized by filtration in a laminar hood in order to prevent CO2 formation in the autoclave. The bottles used for the batch assays were filled with the mineral medium and the pure culture of Thiobacillus denitrificans (10% v/v). Subsequently, the bottles were incubated at 27°C and placed in a gyratory shaker with a shaking speed of 200 rpm. The Thiobacillus denitrificans culture was sub-cultured 4 times prior to being used as seed for the FBRs.
2.2 Start-up and operation of FBRs Two laboratory-scale glass FBRs (volume 580 mL) were used to investigate autotrophic denitrification and denitritation by using thiosulfate as electron donor. The configuration of the FBRs was as reported by Papirio et al. [19]. FBR1 was operated at room temperature (20.0±2.0°C), whereas FBR2 was maintained at 30.0±0.2°C by means of heating wires, a temperature probe and a temperature control system. A quarter of the reactor volume was filled with granular activated carbon (particle size =
6
0.5-1 mm) used as biomass carrier in both FBRs. The FBR recirculation was maintained by a peristaltic pump and adjusted to 25% of carrier fluidization by using a flow rate of 800 mL min-1. Excess thiosulfate compared to the theoretical S2O32- consumption was used to supply sufficient electron donor for complete denitrification and denitritation (Table 2). Initially, both FBRs were filled up with N2-purged mineral medium (Table 3) and activated carbon. The medium was not sterilized and the feed was prepared by using tap water. NaHCO3 was added at a concentration of 1 g L-1and 58 mL of Thiobacillus denitrificans enrichment solution (10% of the entire FBR volume) was injected to both FBR1 and FBR2. The mineral nutrients were added at a 10-fold lower concentration than that used for growing Thiobacillus denitrificans, in order to study the biological nitrate removal at more realistic mineral nutrient levels. During the first experimental period, the FBRs were operated in fed-batch mode for 57 days in order to allow the bacterial colonization of the carrier material. Half of the FBR volume was replaced with fresh medium twice per week and the pH was continuously adjusted to 7.0. From day 58 on, the FBRs were operated in continuous mode. Between day 58 and 182, the FBRs were operated in order to test the effects of HRT, temperature and feed nitrate concentration on denitrification (Table 2, periods I and II). During period I, nitrate and thiosulfate concentrations in the feed were constantly maintained at 1230 and 2260 mg L-1, respectively. HRT was gradually decreased from 24 to 5.4 h by increasing the feed flow rate. In period II (sub-phases 1-3), feed nitrate concentration was gradually decreased from 1230 to 100 mg L-1. In order to maintain the nitrate loading rate constant at 228 mg L-1·h-1, HRT was accordingly decreased down to 0.4 h. In period II (subphases 4-5), HRT was further decreased to 10 and 5 min, keeping the feed nitrate at 100
7
mg L-1. Between period II-4 and 5, HRT was increased back to 5.4 h, in order to stabilize the denitrification process. Between day 184 and 203 (period III), nitrate supplementation was stopped and both reactors were fed with nitrite in order to assess denitritation efficiency (Table 2, period III). HRT was increased back to 5.4 h and feed nitrite was gradually increased from 100 to 1230 mg L-1. Throughout the experimentation, feed thiosulfate was changed according to the feed nitrate or nitrite concentrations, keeping the S2O32-/NO3-and S2O32/NO2- ratios constant. Effluent samples were taken averagely three times a week for nitrate, nitrite, sulfate and thiosulfate analyses.
2.3 Temperature-gradient incubations The effect of different cultivation temperatures on the activity of the FBR cultures was evaluated in a temperature gradient incubator (Terratec®) that was heated at one end and cooled at the other to create a stable temperature gradient. The tested cultivation temperatures ranged from 1 to 46°C with approximately 1.5°C intervals. The incubator was running 2 days prior to the batch assays, in order to stabilize the set temperatures. The temperature in each tube was monitored before and after experimentation by using a thermometer. The 25 mL cultivation tubes were filled with 18 mL of mineral medium (Table 3) prior to purging with N2 for 15 min and sealing with rubber septa. Nitrate and thiosulfate concentrations were 1230 and 2260 mg L-1, respectively. To ensure the desired temperature to the whole cultivation volume, the tubes were incubated in the temperature gradient incubator for 1 h prior to inoculation. After that, 2 mL of stock NaHCO3 solution was added to each tube in order to achieve a concentration of 1 g L-1 and the tubes were inoculated with 2 mL of FBR enrichment cultures (10% v/v). 8
Finally, the tubes were sealed with rubber septa and aluminium caps and incubated in the temperature gradient incubator with a speed of 150 rpm. A culture broth of 0.8 mL was taken every hour during the initial 12 hours from each tube for nitrate, nitrite, sulfate and thiosulfate analyses. The final samples were taken from each tube at 24 h. All the batch assays were carried out in duplicate. The effect of the operating temperatures on the FBR culture activity was modeled by Ratkowsky equation (Equation 4) [23] (Ratkowsky et al., 1983):
= − 1 −
(4)
where g is the generation time, T is the operating temperature (°C) and Tmin and Tmax are the theoretical minimum and maximum temperatures, respectively. The optimal temperature (Topt) was determined as the temperature that resulted in the highest value of 1/. The generation time was calculated as reported in equation 5:
=
(5)
where is the specific growth rate (h-1). The specific growth rate ( ) is usually determined as the slope of the linear part of the plot displaying the natural log of the optical density versus time. In this study, was derived from the nitrate removal rate by determining the slope of the linear part of the plot displaying the evolution of nitrate concentration against time. Finally, the experimental data, obtained at different temperatures from batch assays, were fitted with Ratkowsky equation by using OriginLab software. 9
2.4 Analytical methods The liquid samples from FBRs and batch assays were filtered through 0.45 µm ChromafilXtra PET-20125 membranes (Macherey-Nagel, Germany). Nitrate, nitrite, sulfate and thiosulfate were measured by ion chromatography (IC) as reported by Zou et al. [24]. Effluent pH and dissolved oxygen (DO) were measured directly in the FBRs as reported by Papirio et al. [25]. The volatile solids (VS) concentrations of the biofilmcoated activated carbon were measured at the initial fed-batch operation and during period II on days 151, 167 and 184, as reported by Papirio et al. [19]. The evolution of autotrophic denitrifying microbial communities during FBR operation was carried out by using PCR-DGGE technique. Samples of biofilm-coated activated carbon were taken five times during the experimentation (on days 86, 148, 168, 184 and 204). The biomass samples were sonicated and then the liquid separated from the activated carbon was filtrated with Cyclopore track etched 0.2 µm membranes (Whatman, USA), in order to detach the bacteria from activated carbon. The PCRDGGE analyses were performed as reported by Papirio et al. [19]. The dominant DGGE bands were excised from the gel, eluted in 20 µL of sterile water at 4°C overnight, and amplified by PCR for sequencing. The sequencing was performed by MacroGen (Seoul, Korea) and the obtained data was compared with National Center for Biotechnology Information database to identify the microorganisms.
10
3. Results
3.1 FBR performance 3.1.1 Denitrification Figs. 1 and 2 show the profiles of HRT, nitrate, nitrite, sulfate and pH in both FBR1 (20.0±2.0°C) and FBR2 (30.0±0.2°C) during the over 200 days experimentation. During the initial fed-batch operation, Thiobacillus denitrificans effectively colonized the carrier, resulting in an immobilized VS concentration of 0.2 and 0.17 g mL-1 of activated carbon on day 57 in FBR1 and FBR2, respectively (data not shown). Autotrophic denitrification required 44 days before achieving complete nitrate removal in FBR2 (Fig. 1C). Nitrate removal reached 79% in FBR1 during this experimental phase (Fig. 1C). Thiosulfate oxidation was particularly unstable until day 54 and after which S2O32- remained below the detection limit in both FBRs (Fig. S1B). Effluent pH dropped to 5.7 and 6.7 and sulfate sharply increased to approximately 3700 and 3200 mg L-1 on days 38 and 33 in FBR1 and FBR2, respectively (Fig. 1C and D). During period I, the feed nitrate was 1230 mg/L and the HRT was gradually decreased from 24 to 5.4 h in both FBRs (Fig. 1A). Effluent nitrate and nitrite remained below the detection limit in both FBR1 and FBR2 (Fig. 1B) with a nitrate loading rate of 228 mg·L-1·h-1 (Table 2). Effluent sulfate concentrations remained stable at approximately 3500 mg L-1 in both FBR1 and FBR2 (Fig. 1C), being about 15% higher than the theoretical value of 3065 mg·L-1 which was calculated by the complete feed nitrate reduction to dinitrogen gas based on Equation 3. Thiosulfate was not detected and pH remained at 5.8 in both FBRs (Figs. S1B and 1D). Effluent DO was 0.35 and 0.27 mg L1
on average in FBR1 and FBR2, respectively (Fig. S1A).
11
In period II (sub-phases 1-3), autotrophic denitrification was efficiently performed at 100 mg·L-1 of feed nitrate in both FBR1 and FBR2 at HRT gradually decreasing from 2.6 to 0.4 h, resulting in complete nitrate and nitrite removal and thiosulfate oxidation (Figs. 1B, C and 2B, C). From day 173 to day 182 (period II, sub-phases 4-5), HRT was further decreased in both FBRs first to 10 min (period II-4) and then to 5 min (period II5) obtaining a nitrate loading rate of 600 and 1200 mg·L-1·h-1, respectively, by keeping the feed nitrate at 100 mg·L-1 (Fig. 2A). In period II-4 (days 173-177), nitrate was completely removed except for a peak of 15 mg·L-1 on day 174 in FBR1. No nitrite was observed (Fig. 2B) and sulfate concentrations remained at approximately 275 mg·L-1 (Fig. 2C). In period II-5 (days 181-182), denitrification efficiency was reduced after HRT decrease to 5 min. Effluent nitrate concentrations increased up to 60 and 20 mg·L1
on day 181 and decreased to 27 and 13 mg·L-1 on day 182 in FBR1 and FBR2,
respectively (Fig. 2B), resulting in the nitrate removal efficiency of 60 and 80% on average in FBR1 and FBR2, respectively. Nitrite slightly increased to 4.6 and 2.6 mg·L1
in FBR1, whereas no nitrite was observed in FBR2. Effluent sulfate concentrations
remained at approximately 190 mg·L-1 and 265 mg·L-1 in FBR1 and FBR2, respectively. During this phase, effluent DO continuously increased up to 0.58 mg·L-1 in both FBRs (Fig. 2D). Effluent pH gradually increased from 5.8 (period I) up to approximately 7.0 during period II (Fig. 1D) and thiosulfate remained below the detection limit in both FBRs (Fig. S1B). During period II (days 148-184), VS concentration remained stable at 0.19 and 0.18 g mL-1 of biofilm-coated activated carbon in FBR1 and FBR2, respectively.
12
3.1.2 Denitritation During period III, nitrite was completely removed in both FBRs at feed nitrite 100, 600 and 1230 mg·L-1 at HRT 5.4 h (Fig. 1B). Effluent sulfate concentrations remained at approximately 350, 2000 and 2800 mg·L-1 in period III-1, III-2 and III-3, respectively. This was 75, 65 and 15% higher than the corresponding stoichiometric values (Equation 2), respectively. Thiosulfate was not detected during periods III-1 and III-2, whereas S2O32- concentration increased up to 600 mg·L-1 in period III-3 (Fig. S1B). Effluent pH increased to approximately 7.2 (Fig. 1D) and DO decreased again to 0.35 and 0.27 mg·L-1 in FBR1 and FBR2, respectively (Fig. S1A).
3.2 Microbial community in the FBRs Fig. 3 shows the microbial community profiles of both FBR1 and FBR2 during the experimentation. The numbered bands on the DGGE gel indicate the excised bands that were successfully sequenced. Table 4 reports the evolution of autotrophic denitrifying microbial cultures in both reactors. Results indicate that almost 90% of the sequenced bands represented Thiobacillus denitrificans (Fig. 3 and Table 4). In FBR1 operated at 20.0±2.0°C, Thiobacillus denitrificans and Chryseobacterium hispalense were detected on day 86 (bands 1 and 2, respectively). From day 148 on, only Thiobacillus denitrificans was observed (Table 4 and Fig. 3). In FBR2 operated at 30.0±0.2°C, more microbial species were detected (Table 4) whilst Thiobacillus denitrificans was the dominant species throughout FBR operation. On day 86, besides Thiobacillus denitrificans (band 1), Sediminibacterium salmoneum (bands 3, 4, 5 and 6), Chryseobacterium hispalense (band 7) and Dyella thiooxydans (bands 8 and 9) were
13
observed (Fig. 3 and Table 4). On days 148 and 168, Dyella thiooxydans (bands 10 and 15) was still present, whereas it remained below the DGGE detection on days 184 and 204. In addition, Sediminibacterium salmoneum and Chryseobacterium hispalense faded away from day 148 onwards.
3.3 Modeling of the temperature response by Ratkowsky equation Fig. 4 shows an example of nitrate and nitrite profiles in batch assays at three temperatures (6, 23 and 46°C) that were selected as representatives for all the tested temperatures. At 23°C, nitrate was completely removed in 12 h. However, nitrate consumption was significantly lower at 6 and 46°C, resulting in 25 and 17% of nitrate removal in 12 h and 46 and 26% of nitrate removal in 24 h, respectively. Nitrite accumulation was observed at all temperatures. NO2- increased to approximately 500 mg·L-1 after 12 h at 23°C prior to being completely removed by 24 h. At 6 and 46°C, nitrite accumulated up to 355 and 110 mg·L-1 by 24 h, respectively. Both nitrate and nitrite evolution indicated that complete N removal was not achieved at 6 and 46°C. The experimental data successfully fitted with Ratkowsky equation. Fig. 5 shows the modeling curve and the fitted parameters of b, c, Tmin, Tmax and Topt. The optimal temperature was 26.6°C, in correspondence of the peak of the modeling curve. The minimum (Tmin) and maximum (Tmax) temperatures were estimated as below 1 and 50.8°C, respectively, and determined as the temperature values where √1/ = 0.
14
4. Discussion
This study revealed that autotrophic denitrification in terms of nitrate and nitrite removal can be effectively maintained in two FBRs by using granular activated carbon and thiosulfate as biomass carrier and electron donor, respectively, and a pure culture of Thiobacillus denitrificans as a seed. At the highest feed nitrate of 1230 mg·L-1, nitrate loading rate reached 228 mg·L-1 h-1 (51.5 N-NO3- mg L-1 h-1) which is comparable with the previous studies (Table 5). Sulfate was constantly produced by thiosulfate oxidation and SO42- concentrations were higher than the theoretical ones. This discrepancy was likely due to the observation of other thiosulfate oxidizers (i.e. Dyella thiooxydans) which can utilize dissolved oxygen as an electron acceptor [32], resulting in the oxidation of the excess feed thiosulfate. Thiosulfate-driven autotrophic denitrification produced acidity resulting in a decrease of pH to approximately 5.8 in both FBRs. The external alkalinity supplemented as NaHCO3 (1 g L-1) was not sufficient to maintain neutral conditions in the reactors. In order to reduce 1230 mg·L-1 of nitrate, the stoichiometric amount of NaHCO3 consumed as both carbon source and alkalinity is 1.14 g·L-1and 1.67 g·L-1 according to Mora et al. [10] (Equation 3) and Chung et al. [6], respectively. Also, the oxidation of excess thiosulfate compared to the theoretical S2O32- consumption in the FBRs by other thiosulfate oxidizers (i.e. Dyella thiooxydans) generated acidity and might have consumed part of the supplemented alkalinity. Nevertheless, microbial cultures were effectively adapted to the slightly acidic conditions within the FBRs. To the best of our knowledge, this is the first study reporting continuous-flow autotrophic denitrification at pH 5.8, in contrast with Moon et al. [33] and Koenig and Liu [34] who reported that autotrophic denitrification with elemental sulfur was completely inhibited at pH 6. 15
After operation at high nitrate concentrations in order to achieve high biomass colonization on the carrier, FBRs were operated at feed nitrate of 100 mg·L-1 to evaluate the process robustness under more realistic conditions for nitrate contaminated wastewaters. Efficient denitrification was maintained even at HRT of 10 min, which has never been reported previously (Table 5). The nitrate loading rate reached the highest of 600 mg·L-1 h-1 (Table 5, 135.4 N-NO3- mg L-1 h-1). The detection of residual nitrate and nitrite in the effluent at a HRT of 5 min indicated that FBRs approached the maximum loading capacity, as Sierra-Alvarez et al. [18] reported that nitrate or nitrite accumulation is a reliable marker of reactor overloading. This high denitrification efficiency was due to the effective mass transfer of the substrates to microorganisms of the FBRs [21]. At feed nitrate of 100 mg L-1, sulfate production was still higher than the stoichiometric value due to the excess of thiosulfate in the feed and the higher DO levels caused by the increase of the feed flow rate. Effluent pH increased to approximately 7.0 due to decreased acidity production during this phase, likely providing favorable conditions for autotrophic denitrification at such short HRTs [34]. The FBR biofilm cultures also demonstrated to efficiently remove nitrite when it was used as the sole electron acceptor, as previously reported by Sun and Nemati [7] and Zhou et al. [16]. In the batch assays, nitrite accumulated as an intermediate of denitrification at all temperatures. This was due to the delay in the activation of the nitrite-reducing enzymes or the lower specific nitrite utilization rate [10]. When nitrate was fed to the reactors, nitrite was below the detection limit except when FBRs were operated at the maximum nitrate loading. FBRs permitted an efficient contact between the microorganisms and substrate that allowed a faster removal of nitrite.
16
During period III, nitrite was fed to both reactors, resulting in complete nitrite removal at HRT 5.4 h. Nitrite loading rate reached the highest of 228 mg L-1 h-1 which was comparable with nitrate loading rate during denitrification. Autotrophic denitritation consumed acidity (Equation 2) that resulted in the increase of effluent pH to 7.2, in agreement with Chung et al. [6]. The S2O32-/NO2- ratio used was much higher than the theoretical ratio of 0.5 mol/mol (Equation 2) and resulted in 600 mg L-1 of effluent thiosulfate at feed nitrite of 1230 mg L-1. Temperature has been shown to significantly affect the performance of autotrophic denitrification in pilot and full scale applications [15, 16]. In the present study, FBR denitrification with thiosulfate was efficiently maintained at both 20±2 and 30±0.2°C. Some differences in the behaviors of the two reactors were observed during the initial fed-batch operation and at the lowest HRTs used (10 and 5 min), demonstrating that the process was more favorable at 30°C. The optimal temperature of nitrate removal obtained by Ratkowsky modeling (26.6°C) also confirmed that denitrification efficiency was the highest at temperatures close to 28-32°C as reported by Kelly and Wood [14]. Furthermore, the fact that the optimal temperature (26.6°C) was close to the operating temperatures of both FBRs accounts for the comparable denitrification performances of FBR1 and FBR2. The minimum and maximum growth temperatures (Tmin = below 1°C and Tmax = 50.8°C) for Thiobacillus denitrificans dominated culture were also obtained in this study and have not been previously reported elsewhere. In contrast, Oh et al. [17] demonstrated that denitrification was completely inhibited at 40°C by using a mixed autotrophic denitrifying culture. To our knowledge, the temperature dependency of nitrate removal was for the first time thoroughly determined in this study for an autotrophic denitrifying culture. Ratkowsky
17
equation has been previously applied to model the effect of temperature on iron and sulfur oxidizing microorganisms [35]. In that study, the specific growth rate (µ) (Equation 5) was derived from Fe2+ oxidation or SO42- production rates due to the largely proportional relationship between bacteria growth rate and substrate utilization or product formation rates. Similarly, the growth rate of autotrophic denitrifying organisms has been reported to be largely proportional to nitrate utilization rate [17, 36]. Therefore, µ in Ratkowsky equation was considered as the nitrate removal rate. Nevertheless, autotrophic denitrification contains two steps in which nitrate is first reduced to nitrite and then to dinitrogen gas. This study demonstrated the effect of temperature range on nitrate removal rate of autotrophic denitrification by Ratkowsky model whilst the effect of temperature range on nitrite removal rate will require further study. The evolution of microbial communities in the FBRs revealed the dominance of Thiobacillus denitrificans even after 204 days operation with non-sterilized growth medium. This species demonstrated to be able to reduce both nitrate and nitrite under chemolithotrophic conditions with thiosulfate as electron donor. The initial presence of Sediminibacterium salmoneum [37], Chryseobacterium hispalense [38] and Dyella thiooxydans [32] was due to bacterial contamination of the open FBR systems. In contrast with Moon et al. [33], sulfate reducing bacteria were not detected despite the presence of high sulfate concentrations and the possible organic debris from bacterial lyses.
18
5. Conclusions High-rate, high-efficiency autotrophic denitrification was achieved in fluidized-bed biofilms. The following specific conclusions can be drawn for this study: 1) Autotrophic denitrification and denitritation with thiosulfate as electron donor were effectively maintained in two FBRs operated at 20.0±2.0 and 30.0±0.2°C. The highest nitrate and nitrite loading rates were 600 and 228 mg L-1·h-1, respectively. 2) Autotrophic denitrification was successfully operated even at HRT 10 min, resulting in complete nitrate and nitrite removal. 3) The microbial communities of both FBRs inoculated with a pure culture of Thiobacillus denitrificans were dominated by T. denitrificans even after 204 days operation with non-sterilized growth medium and tolerated pHs as low as 5.8. 4) Ratkowsky model was successfully used for the first time to simulate the effect of temperature on the nitrate removal efficiency of Thiobacillus denitrificans dominated biofilm. The minimum, optimal and maximum temperatures according to the model were below 1, 26.6 and 50.8°C, respectively.
19
6. References [1] A. Bhatnagar, E. Kumar, M. Sillanpää, Nitrate removal from water by nanoalumina: Characterization and sorption studies, Chem. Eng. J. 163 (2010) 317-323.
[2] J.Y. Park and Y.J. Yoo, Biological nitrate removal in industrial wastewater treatment: which electron donor we can choose, Appl. Microbiol. Biotechnol. 82 (2009) 415-429.
[3] C. Chow and C. Hong, Dietary vitamin E and selenium and toxicity of nitrite and nitrate, Toxicology 180 (2002) 195-207.
[4] L. Knobeloch, B. Salna, A. Hogan, J. Postle, H. Anderson, Blue babies and nitratecontaminated well water, Environ. Health Perspect. 108 (2000) 675-678.
[5] F. Di Capua, S. Papirio, P.N.L. Lens, G. Esposito, Chemolithotrophic denitrification in biofilm reactors, Chem. Eng. J. 280 (2015) 643-657.
[6] J. Chung, K. Amin, S. Kim, S. Yoon, K. Kwon, W. Bae, Autotrophic denitrification of nitrate and nitrite using thiosulfate as an electron donor, Water Res. 58 (2014) 169178.
[7] Y. Sun and M. Nemati, Evaluation of sulfur-based autotrophic denitrification and denitritation for biological removal of nitrate and nitrite from contaminated waters, Bioresour. Technol. 114 (2012) 207-216.
[8] K. Tang, S. An, M. Nemati, Evaluation of autotrophic and heterotrophic processes in biofilm reactors used for removal of sulphide, nitrate and COD, Bioresour. Technol. 101 (2010) 8109-8118.
20
[9] E. Sahinkaya, N. Dursun, A. Kilic, S. Demirel, S. Uyanik, O. Cinar, Simultaneous heterotrophic and sulfur-oxidizing autotrophic denitrification process for drinking water treatment: control of sulfate production, Water Res. 45 (2011) 6661-6667.
[10] M. Mora, A. Guisasola, X. Gamisans, D. Gabriel, Examining thiosulfate-driven autotrophic denitrification through respirometry, Chemosphere 113 (2014) 1-8.
[11] C. Fajardo, M. Mora, I. Fernández, A. Mosquera-Corral, J. L. Campos, R. Méndez, Cross effect of temperature, pH and free ammonia on autotrophic denitrification process with sulphide as electron donor, Chemosphere 97 (2014) 10-15.
[12] B. F. Taylor and D. S. Hoare, Thiobacillus denitrificans as an obligate chemolithotroph, Arch. Microbiol. 80 (1971) 262-276.
[13] A. T. Hoor, A new type of thiosulphate oxidizing, nitrate reducing microorganism: Thiomicrospira denitrificans sp. Nov., Neth. J. Sea Res. 9 (1975) 344-350.
[14] D.P. Kelly and A.P. Wood, Confirmation of Thiobacillus denitrificans as a species of the genus Thiobacillus, in the beta-subclass of the Proteobacteria, with strain NCIMB 9548 as the type strain, Int. J. Syst. Evol. Microbiol. 50 (2000) 547-550.
[15] E. Sahinkaya, A. Kilic, B. Duygulu, Pilot and full scale applications of sulfur-based autotrophic denitrification process for nitrate removal from activated sludge process effluent, Water Res. 60 (2014) 210-217.
[16] W. Zhou, Y. Sun, B. Wu, Y. Zhang, M. Huang, T. Miyanaga, Z. Zhang, Autotrophic denitrification for nitrate and nitrite removal using sulfur-limestone, J. Environ. Sci. 23 (2011) 1761-1769.
21
[17] S. Oh, K. Kim, H. Choi, J. Cho, I. Kim, Kinetics and physiological characteristics of autotrophic denitrification by denitrifying sulfur bacteria, Water Sci. Technol. 42 (2000) 59-68.
[18] R. Sierra-Alvarez, R. Beristain-Cardoso, M. Salazar, J. Gómez, E. Razo-Flores, J. A. Field, Chemolithotrophic denitrification with elemental sulfur for groundwater treatment, Water Res. 41 (2007) 1253-1262.
[19] S. Papirio, A. Ylinen, G. Zou, M. Peltola, G. Esposito, J. A. Puhakka, Fluidizedbed denitrification for mine waters. Part I: low pH and temperature operation, Biodegradation 25 (2014) 425-435.
[20] G. Zou, S. Papirio,, E. Van Hullebusch, J.A. Puhakka, Fluidized-bed denitrification of mining water tolerates high nickel concentrations, Bioresour. Technol. 179 (2015) 284-290.
[21] H. Kim, I. Lee, J. Bae, Performance of a sulphur-utilizing fluidized bed reactor for post-denitrification, Process Biochem. 39 (2004) 1591-1597.
[22] M. Soares, Denitrification of groundwater with elemental sulfur, Water Res. 36 (2002) 1392-1395.
[23] D.A. Ratkowsky, R.K. Lowry, T.A. McMeekin, A.N. Stokes, R.E. Chandler, Model for bacterial culture growth rate throughout the entire biokinetic temperature range, J. Bacteriol. 154 (1983) 1222-1226.
[24] G. Zou, S. Papirio, A. Ylinen, F. Di Capua, A-M. Lakaniemi, J.A. Puhakka, Fluidized-bed denitrification for mine waters. Part II: effects of Ni and Co, Biodegradation 25 (2014) 417-423. 22
[25] S. Papirio, G. Zou, A. Ylinen, F. Di Capua, F. Pirozzi, J.A. Puhakka, Effect of arsenic on nitrification of simulated mining water, Bioresour. Technol. 164 (2014) 149154.
[26] E. Sahinkaya, A. Kilic, Heterotrophic and elemental-sulfur-based autotrophic denitrification processes for simultaneous nitrate and Cr(VI) reduction, Water Res. 50 (2014) 278-286.
[27] H.S. Moon, K. Nam, J.Y. Kim, A long-term performance test on an autotrophic denitrification column for application as a permeable reactive barrier, Chemosphere 73 (2008) 723-728. [28] J. Gu, W. Qiu, A. Koenig, Y. Fan, Removal of high NO3- concentrations in saline water through autotrophic denitrification by the bacterium Thiobacillus denitrificans strain MP, Water Sci. Technol. 49 (2004) 105-112.
[29] A. Koenig, L. Liu, Kinetic model of autotrophic denitrification in sulphur packedbed reactors, Water Res. 35 (2001) 1969-1978.
[30] S. Oh, Y. Yoo, J. Young, I. Kim, Effect of organics on sulfur-utilizing autotrophic denitrification under mixotrophic conditions, J. Biotechnol. 92 (2001) 1-8.
[31] E. Sahinkaya, N. Dursun, Use of elemental sulfur and thiosulfate as electron donor for water denitrification, Bioprocess Biosyst. Eng. 38 (2015) 531-541.
[32] R. Anandham, S-W. Kwon, P.I. Gandhi, S-J. Kim, H-Y. Weon, Y-S. Kim, T-M. Sa, Y-K. Kim, H-J. Jee, Dyella thiooxydans sp. nov., a facultatively chemolithotrophic, thiosulfate-oxidizing bacterium isolated from rhizosphere soil of sunflower (Helianthus annuus L.), Int. J. Syst. Evol. Microbiol. 61 (2011) 392-398. 23
[33] H.S. Moon, K-H. Ahn, S. Lee, K. Nam, J.Y. Kim, Use of autotrophic sulfuroxidizers to remove nitrate from bank filtrate in a permeable reactive barrier system, Environ. Pollut. 129 (2004) 499-507.
[34] A. Koenig and L. Liu, Use of limestone for pH control in autotrophic denitrification: continuous flow experiments in pilot-scale packed bed reactors, J. Biotechnol. 99 (2002) 161-171.
[35] P. Franzmann, C. Haddad, R. Hawkes, W. Robertson, J. Plumb, Effects of temperature on the rates of iron and sulfur oxidation by selected bioleaching Bacteria and Archaea: Application of the Ratkowsky equation, Miner. Eng. 18 (2005) 13041314.
[36] H. Zeng and T.C. Zhang, Evaluation of kinetic parameters of a sulfur–limestone autotrophic denitrification biofilm process, Water Res. 39 (2005) 4941-4952.
[37] J.H. Qu and H.L. Yuan, Sediminibacterium salmoneum gen. nov., sp. nov., a member of the phylum Bacteroidetes isolated from sediment of a eutrophic reservoir, Int. J. Syst. Evol. Microbiol. 58 (2008) 2191-2194.
[38] M.C. Montero-Calasanz, M. Goker, M. Rohde, C. Sproer, P. Schumann, H.J. Busse, M. Schmid, B.J. Tindall, H.P. Klenk, M. Camacho, Chryseobacterium hispalense sp. nov., a plant-growth-promoting bacterium isolated from a rainwater pond in an olive plant nursery, and emended descriptions of Chryseobacterium defluvii, Chryseobacterium indologenes, Chryseobacterium wanjuense and Chryseobacterium gregarium, Int. J. Syst. Evol. Microbiol. 63 (2013) 4386-4395.
24
Figures Figure 1- The profiles of HRT and effluent nitrate, nitrite, sulfate concentrations and pH throughout the FBR1 and FBR2 operation. The theoretical sulfate concentration was calculated based on the complete feed nitrate and nitrite reduction to dinitrogen gas according to equation 1 and 3, respectively. Figure 2- The profiles of HRT and effluent nitrate, nitrite, sulfate and DO concentrations in the period II-3, 4 and 5 of FBR1 and FBR2 operations. The theoretical sulfate concentration was calculated based on the complete feed nitrate reduction to dinitrogen gas according equation 1. Figure 3 - Bacterial community profiles in FBR1 and FBR2 on days 86, 148, 168, 184 and 204. Figure 4 - Nitrate and nitrite evolution in batch assays at three temperatures 6, 23 and 46°C. Figure 5 - The modeling curve by Ratkowsky equation showing the relationship of temperature to nitrate removal in autotrophic denitrification. Figure S1- The profiles of effluent DO and thiosulfate throughout FBR1 and FBR2 operations.
25
26
27
28
29
30
Table 1 - The composition of the medium used for activation of Thiobacillus denitrificans. Solution A
Concentration
KH2PO4
2.0 g L-1
KNO3
2.0 g L-1
NH4Cl
1.0 g L-1
MgSO4×7H2O
0.8 g L-1
Trace elements
*
pH adjusted to 7.0 with NaOH Solution B Na2S2O3×5H2O
5 g L-1
Solution C NaHCO3
1.0 g L-1
Solution D FeSO4×7H2O
2.0 mg L-1
H2SO4 (0.1 N)
1.0 mL L-1
*The concentrations of trace elements were as presented in the text (section 2.1).
31
Table 2 - Operational conditions and feed composition of FBR1 and FBR2 at different experimental periods.
2260
NO3- or NO2loading rate (mg L-1·h-1) 51
S2O32- /NO3or NO2- ratio (mol/mol) 1.0
0
2260
77
1.0
1230
0
2260
123
1.0
6.4
1230
0
2260
192
1.0
141 − 147
5.4
1230
0
2260
228
1.0
II-1
148 − 158
2.6
600
0
1105
228
1.0
II-2
159 − 165
0.9
200
0
368
228
1.0
II-3
166 − 172
0.4
100
0
184
228
1.0
II-4
173 − 177
0.17
100
0
184
600
1.0
II-5
181 − 182
0.08
100
0
184
1200
1.0
III-1
184 − 189
5.4
0
100
219
19
0.9
III-2
190 − 196
5.4
0
600
1314
111
0.9
III-3
197 − 204
5.4
0
1230
2694
228
0.9
Period
Time (days)
HRT (h)
NO3(mg L-1)
NO2(mg L-1)
S2O32(mg L-1)
I-1
58 − 71
24
1230
0
I-2
72 − 85
16
1230
I-3
86 − 123
10
I-4
124 − 140
I-5
32
Table 3 - The composition of the mineral medium used in the FBRs. Compound
Concentration (g L-1)
KH2PO4
0.2
NH4Cl
0.1
MgSO4×7H2O
0.08
Trace elements
*
FeSO4×7H2O
2 mg L-1
KNO3
2
Na2S2O3×5H2O
5
NaHCO3
1 pH was adjusted to 7.0
*The concentrations of trace elements were 10 times lower than that used for activating Thiobacillus denitrificans, as explained in section 2.1.
33
Table 4 - The identity of the sequenced DGGE bands (16SrDNA) based on the biomass samples taken from FBR1 and FBR2. FBRs
Similarity (%)
Phylogenetic group (class/order/family)
Thiobacillus denitrificans
Accession Number NR_025358.1
100
β/Hydrogenophilales/Hydrogenophilaceae
2
Chryseobacterium hispalense
KM199279.1
100
Flavobaceriia/Flavobacteriales/Flavobacteriaceae
148
1
Thiobacillus denitrificans
NR_025358.1
100
β/Hydrogenophilales/Hydrogenophilaceae
168
1,11
Thiobacillus denitrificans
NR_025358.1
99.8-100
β/Hydrogenophilales/Hydrogenophilaceae
184
16,17,18,19,20
Thiobacillus denitrificans
NR_025358.1
99.5-100
β/Hydrogenophilales/Hydrogenophilaceae
204
25, 26, 27,28
Thiobacillus denitrificans
NR_025358.1
99.8-100
β/Hydrogenophilales/Hydrogenophilaceae
1
Thiobacillus denitrificans
NR_025358.1
100
β/Hydrogenophilales/Hydrogenophilaceae
KF358456.1
99.8-100
7
Sediminibacterium salmoneum Chryseobacterium hispalense
KM199279.1
100
Sphingobacteriia/Sphingobacteriales/Chitinophagac eae Flavobaceriia/Flavobacteriales/Flavobacteriaceae
8,9
Dyella thiooxydans
NR_116006.1
99.8-100
Flavobaceriia/Flavobacteriales/Flavobacteriaceae
1
Thiobacillus denitrificans
NR_025358.1
100
β/Hydrogenophilales/Hydrogenophilaceae
10 12,13,14
Dyella thiooxydans
NR_116006.1
99.8-100
Flavobaceriia/Flavobacteriales/Flavobacteriaceae
Thiobacillus denitrificans
NR_025358.1
100
β/Hydrogenophilales/Hydrogenophilaceae
15
Dyella thiooxydans
NR_116006.1
99.6
Flavobaceriia/Flavobacteriales/Flavobacteriaceae
184
16,21,22,23,24
Thiobacillus denitrificans
NR_025358.1
99.3-100
β/Hydrogenophilales/Hydrogenophilaceae
204
29,30,31
Thiobacillus denitrificans
NR_025358.1
99.1-100
β/Hydrogenophilales/Hydrogenophilaceae
Operation day
Band label
Closest species in GenBank database
1 86 FBR1 (20.0±2.0°C)
86
FBR2 (30.0±0.2°C)
148 168
3,4,5,6
Table 5 Performance of biofilm reactors for sulfur-based autotrophic denitrification as reported in literature.
Culture
Feed N (mg NNO3-/L)
T (°C)
Electron donor
HRT (h)
N-NO3removal rate (mg N-NO3-/ L h)
28-30
S0
31.2-
2.8-4.3
[26]
13-26
0
1.3-10.0
[15]
References
Packed-bed reactor (PBR) Activated sludge
50-75
12.0 Denitrifying
30-60
S
3.0-6.0
activated sludge Mixed culture
(pilot scale) 0
144-748
23-25
S
0.4-3.0
245.0-338.8
[7]
10-100
5-32
S0
3.0-6.0
3.3-13.3
[16]
Enrichment from activated sludge
60
20
S0
24.048.0
1.3-2.5
[27]
Enrichment from
18-102
30±2
S0
1.8-30.5
10.1-12.5
[18]
250
22±2
S0
14.3-
17.1
[28]
60-251
20-25
0
S
5.5-8.9
11.3-36.3
[34]
60-400
20-25
S0
4.0-14.0
20.0-32.1
[29]
100
20-25
S0
7.1-1.5
14.1-60.0
[30]
28-30
S0
5.0
2.9-8.6
[31]
0.2-6.1
109.4-147.5
[21]
0.1-5.4
51.5-135.4
Present study
from oil reservoir Municipal activated sludge
anaerobic sludge Thiobacillus
30.5
denitrificans Thiobacillus denitrificans Thiobacillus denitrificans Activated sludge
Fluidized-bed reactor (FBR) Activated sludge Enrichment from
25-75 20-700
20
23-278
20±2;
0
S
/S2O32-
a tidal flat Thiobacillus denitrificans
S2O32-
30
35
High rate autotrophic denitrification in fluidized-bed biofilm reactors G. Zou, S. Papirio, A-M. Lakaniemi, S.H. Ahoranta, J.A. Puhakka
7/9/2015
HIGHLIGHTS -
High rate, high efficiency autotrophic denitrification was demonstrated in a FBR
-
Complete nitrate and nitrite removal was achieved at HRTs as low as 10 min
-
The nitrate and nitrite loading rates reached 600 and 228 mg·L-1·h-1, respectively
-
The biofilms were dominated by T. denitrificans and tolerated pHs as low as 5.8
-
The optimal temperature for nitrate removal was 26.6°C based on Ratkowsky modeling
36