Bioresource Technology 187 (2015) 30–36
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Activation of accumulated nitrite reduction by immobilized Pseudomonas stutzeri T13 during aerobic denitrification Fang Ma, Yilu Sun, Ang Li ⇑, Xuening Zhang, Jixian Yang State Key Laboratory of Urban Water Resource and Environment, Harbin Institute of Technology, Harbin 150090, People’s Republic of China
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
Reduction of accumulated nitrite
could be promoted by immobilization. A high total nitrogen (TN) removal could be achieved in immobilization systems. The performance of mycelial pellets and polyurethane foam cubes system were steady.
a r t i c l e
i n f o
Article history: Received 13 February 2015 Received in revised form 10 March 2015 Accepted 11 March 2015 Available online 24 March 2015 Keywords: Aerobic denitrification Accumulation of nitrite TN removal Immobilization Pseudomonas stutzeri T13
a b s t r a c t The excellent removal efficiency of nitrate by the aerobic denitrifier, Pseudomonas stutzeri T13, was achieved in free cells system. However, poor nitrite reduction prevents efficient aerobic denitrification because of the nitrite accumulation. This problem could be conquered by immobilizing the cells on supports. In this study, strain T13 was immobilized by mycelial pellets (MPs), polyurethane foam cubes (PFCs) and sodium alginate beads (SABs). Higher removal percentages of TN in MP (43.78%), PFC (42.31%) and SAB (57.25%) systems were achieved compared with the free cell system (29.7%). Furthermore, the optimal condition for immobilized cell systems was as follows: 30 °C, 100 rpm shaking speed and pH 7. The shock-resistance of SAB system was relatively poor, which could collapse under either alkaline (pH = 9) or high rotating (200 rpm) conditions. The recycling experiments demonstrated that the high steady TN removal rate could be maintained for seven cycles in both MP and PFC systems. Ó 2015 Elsevier Ltd. All rights reserved.
1. Introduction Aerobic denitrification as a novel biological technology of nitrogen removal that could successfully overcome the incompatibility of nitrification with denitrification caused by different oxygen demands has gained a great of attention in recent years (Zhu et al., 2008). Since the first microbe with special function of ⇑ Corresponding author at: State Key Laboratory of Urban Water Resource and Environment, School of Municipal and Environmental Engineering, Harbin Institute of Technology, Harbin 150090, People’s Republic of China. Tel./fax: +86 451 86283787. E-mail address: [email protected] (A. Li). http://dx.doi.org/10.1016/j.biortech.2015.03.060 0960-8524/Ó 2015 Elsevier Ltd. All rights reserved.
reducing nitrate to gaseous nitrogen aerobically has been found (Robertson et al., 1985), some novel aerobic denitrifiers have been isolated and reported, such as Thauera mechernichensis sp. nov. TL1T (Scholten et al., 1999), Pseudmonas stutzeri YZN-001 (Zhang et al., 2011a) and Klebsiella pneumonia CF-S9 (Padhi et al., 2013). Aerobic denitrifiers could conduct an aerobic respiratory process, in which nitrate is gradually reduced to N2 (Ji et al., 2013). The periplasmic nitrate reductase (Nap) located on the periplasmic side of the cell membrane of the aerobic denitrifier could guarantee the slight inhibition of nitrate reduction by dissolved oxygen (DO) (Sparacino-Watkins et al., 2014). However, nitrite reductases (Nir) in aerobic denitrification bacteria are still sensitive to oxygen,
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similar to traditional denitrifiers (Körner and Zumft, 1989). In our previous report, reduction of nitrite has been proven to be the ratelimiting step during aerobic denitrification, especially under high nitrate loading condition, which could be improved by adjusting the DO to a relatively low level (Sun et al., 2015).In addition, accumulation of nitrite as the end-product instead of gaseous nitrogen from the nitrate reduction process would decrease the removal efficiency of total nitrogen (TN) severely (Körner and Zumft, 1989; Liang et al., 2011). Therefore, effective methods to break this bottleneck and lay the foundation for further practical application of aerobic denitrification are necessary. Microbial immobilized technology has been widely used in biological treatment of contaminants (Isaka et al., 2012; Nair et al., 2007; Shi et al., 2014; Zhang et al., 2011b). The limited O2 transfer rate as a defect of immobilized cells could be regarded as an advantage from a certain perspective. The gradient concentration of dissolved oxygen in the inner spaces of the supports would provide an effective area for accumulated nitrite reduction during aerobic denitrification. In this study, three kinds of supports, namely, mycelial pellets (MPs), polyurethane foam cubes (PFCs) and sodium alginate beads (SABs), were used for immobilizing strain T13 to improve the aerobic denitrification TN removal performance. The nitrogen removal efficiency and the accumulation of nitrite in free cell and immobilized cell systems were compared. The results provided an efficient method to activate the reduction of nitrite accumulating during aerobic denitrification and improve TN removal. 2. Methods 2.1. Strains and media P. stutzeri T13 and Aspergillus niger Y3 were provided by the State Key Laboratory of Urban Water Resource and Environment, Harbin Institute of Technology, China. In previous study, strain T13 was confirmed to be an aerobic denitrification bacterium and exhibited an excellent performance on nitrate reduction under aerobic condition (Li et al., 2012; Sun et al., 2015). Strain Y3 was used to flocculate into the mycelial pellet under the submerged shaking condition. The denitrification medium (DM) used for T13 strain cultivation contained (per litter of distilled water) 4.7 g of sodium succinate, 1.5 g of KNO3, 1.5 g of KH2PO4, 7.9 g of Na2HPO4, 0.5 g of MgSO47H2O and 1 ml of a trace element solution, with pH 7.0– 7.2. The trace element solution contained (per litter of distilled water) 50 g of EDTA, 2.2 g of ZnSO4, 5.5 g of CaCl2, 5.06 g of MnCl24H2O, 5.0 g of FeSO47H2O, 1.1 g of (NH4)6Mo7O24H2O, 1.57 g of CuSO45H2O and 1.61 g of CoCl26H2O.The liquid medium (LM) for culturing strain Y3 into mycelial pellet contained (per litter of distilled water) 10 g of glucose, 1.0 g of KH2PO4, 1.0 g of NH4Cl and 0.5 g of MgSO4 (Zhang et al., 2011b). 2.2. Preparation of mycelial pellet and polyurethane foam The spores of strain Y3 on the solid medium were washed into a 100 ml flask containing several glass beads with the distilled water under sterile condition. The spore suspension was obtained after gentle shaking and the absorbance was measured with a visible spectrophotometer under the wavelength of 620 nm to quantify the spore concentration (Van Suijdam et al., 1980; Van Suijdam et al., 1982). The spore suspension of strain Y3 was then inoculated into LM to cultivate the mycelial pellet at 30 °C and 140 rpm. The inoculation quantity was calculated as follows:
Volume of spore suspension for inoculation ¼
0:25 0:1% volume of liquid medium absorbance of spore suspension ðAÞ
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The polyurethane foam was cut into approximately 0.5 cm cubes, washed three times with distilled water, and then autoclaved (121 °C, 30 min) before used. The average dry weight of MPs and PFCs were measured. 2.3. Preparation of free and immobilized cells Strain T13, grown in DM for 24 h was harvested by centrifugation at 10,000 rpm for 5 min. The cells were suspended in sterile water at a cell concentration of 1 OD660, and the dry weight of 1 ml suspension was determined to quantify the biomass. A certain amount of homogeneous MPs and PFCs were added into two 1 L flasks containing 200 mL of DM, and then inoculated with 10 mL cell suspension. The flasks were incubated at 30 °C on a shaker at 160 rpm for 24 h (one cycle). Afterwards, the supernatant was thrown out and fresh DM was added instead. Immobilization could be finished after 4–5 cycles, as a large number of biomass was adsorbed visibly in the inside and outside the MPs (Fig. S1A) and PFCs (Fig. S1B). The average dry weights of the carriers after immobilization were measured. The quantity of biomass immobilized by either per mycelial pellet or polyurethane foam cube was calculated as follows: The average biomass immobilized by per carrier ðgÞ ¼
dry weight after immobilization ðgÞ dry weight before immobilization ðgÞ number of carriers
The sodium alginate and powder-activated carbon were mixed at the dry weight ratio of 8 (wt/wt) and dissolved in the distilled water to compound the 4% sodium alginate solution. The autoclaved solution and T13 cell suspension at a cell concentration of 1 OD660 were mixed. The mixture was extruded through a syringe into 4% CaCl2 solution and solidified for 24 h at 4 °C to obtain the alginate entrapped cells (Fig. S1C). After immobilization, the cells adsorbed by three different carriers were probed by scanning electron microscopy (SEM). 2.4. Adsorption abilities of nitrate and nitrite in different carriers The same quality (5 g) of the three kinds of carriers (MPs, PFCs and SABs) without cells were added in triplicate into 250 mL flasks each containing 100 mL of DM. A medium without additive was used as control. The nitrate concentration was regularly determined to estimate the adsorption of nitrate nitrogen by the carriers. The adsorption of carriers on nitrite nitrogen was conducted in DM containing nitrite instead of nitrate. All experiments were performed in triplicate. In this part, for accurate measurement, the concentrations of nitrate and nitrite were determined by ion chromatography. 2.5. Aerobic denitrification performance of free and immobilized cells To investigate the effect of different immobilization carriers on the aerobic denitrification performance of strain T13, the free cells (cell suspension of T13) and immobilized cells (MPs, PFCs and SABs) were inoculated into four 250 mL shaking flasks each containing 100 mL of DM. The inoculum biomass was approximately equivalent to the control by calculating the dry weight of the biomass at different conditions (Table S1). A medium without inoculation was used as control. All flasks were covered with gaspermeable seals to provide an aerobic condition to the systems, and then cultivated with constant shaking at 160 rpm and 30 °C for 24 h. The samples were obtained from the flasks at 3-h intervals to determine the optical density at 660 nm (OD660), as well as the concentrations of nitrate nitrogen (NO 3 -N), nitrite nitrogen (NO2 N) and total nitrogen (TN). Each experiment was performed in triplicate.
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2.6. Impact of immobilization on the nitrite reduction ability of strain T13 To evaluate the effect of immobilization on nitrite reduction of strain T13 during aerobic denitrification, the equivalent biomass of the free cells and the three different kinds of immobilized cells were inoculated in four 250 mL flasks with 100 mL of DM containing nitrite instead of nitrate. A non-inoculated medium was used for comparison. The flasks were cultivated aerobically at 160 rpm and 30 °C for 24 h. The samples were regularly collected at 3-h intervals to measure the optical density at 660 nm (OD660), as well as the concentration of nitrite nitrogen (NO 2 -N) and the removal percentage of nitrite was calculated. Each experiment was performed in triplicate.
hand, a large number of biomass was observed in the inner spaces of PFCs. For SABs, the colour of the carriers changed from glossy black to grey during immobilization. Thus, colour changes could be distinguished through macroscopic observation between the carriers with cells and the pure ones. After immobilization, the MPs, PFCs and SABs were analysed by SEM (Fig. S2). A large amount of biomass was accessibly immobilized by all kinds of carriers. Although the internal structure of the polyurethane foam was not as compact as the other two carriers, sufficient biomass was guaranteed by the thick biofilms (Fig. S2B). Moreover, the biomass located inside the carriers were observed more than the biomass on the surface. It demonstrated that the strain T13 tended to grow at relatively low dissolved oxygen circumstance which could be provided inside the carriers through mass transfer limitation.
2.7. Stress tests of the immobilized systems Batch experiments were conducted to examine the effects of temperature, pH and shaking speed on TN removal of the different immobilized systems. In the first set of experiments, the cultivation temperatures were 10, 20, 25, 30 and 35 °C, and the experiments were conducted at initial pH of 7 and shaking speed of 160 rpm. In the second set of experiments, the initial pH value was adjusted to 5, 6, 7, 8 and 9, and the experiment was conducted at 30 °C and shaking speed of 160 rpm. In the third set of experiments, the shaking speed was controlled at 0, 50, 100, 160 and 200 rpm, and the experiment was carried out at initial pH of 7 and 30 °C. In each batch, the sample was obtained at 18 h and the removal of TN was analysed. All the experiments were performed in triplicate.
3.2. Adsorption abilities for nitrate and nitrite by different carriers The adsorption abilities for nitrate and nitrite by different carrier materials were investigated, the results are shown in Fig. 1. Differences in the adsorption abilities on nitrate (Fig. 1A) or nitrite (Fig.1B) between the three carrier materials were observed. For the adsorption of nitrate, SABs performed the best adsorption capacity among the three carriers, with saturated adsorption of 10.71 mg/L. The NO 3 -N in the medium was adsorbed rapidly during the first 3 h and remained constant. Meanwhile, the approximately 3 h of saturated adsorption time (SAT) of SABs was the shortest among the other two materials. By contrast, the specific maximum adsorption of MPs and PFCs for nitrate was 7.28 and 5.32 mg/L
2.8. Reuse of immobilized cells on aerobic denitrification
2.9. Analytical methods of water quality
3. Results and discussion 3.1. Morphological characteristics of the carriers after immobilization In the process of immobilization, for MPs and PFCs, the cells of strain T13 were immobilized by adsorption firstly. Afterwards, the biomass on carriers was mainly enlarged by propagation. Both kinds of carriers after immobilization seemed obviously different from the ones without cells. On one hand, the colour of MPs before immobilization was white, but changed to beige after immobilization, similar to that of the strain T13 colony (Fig. S1A). On the other
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The concentrations of ammonium, nitrate, nitrite and TN were determined according to standard methods, i.e., by Nessler’s reagent spectrophotometry, phenol disulfonic acid photometry, N-(1-naphthyl)-ethylene diamine photometry and alkaline potassium persulfate photometry, respectively (APHA, 2012). The growth of T13 was measured using a spectrophotometer at a wavelength of 660 nm.
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Cells immobilized by mycelial pellets Cells immobilized by polyurethane foam cubes Cells immobilized by sodium alginate beads
12 Adsorption of NO3--N (mg/L)
The recycling experiments using the three kinds of immobilized cells to reduce nitrate during aerobic denitrification in three 250 mL flasks containing 100 mL of DM were carried out. The experiments were conducted at initial pH of 7, shaking speed of 100 rpm and 30 °C. After each batch, the immobilization materials were collected in a clean bench and washed three times with sterile water to remove the free cells. Afterwards, the immobilized cells were added separately into the fresh DM for the next cycle. Each cycle lasted for 18 h. At the end of each cycle, the OD660 value and removal of TN were examined.
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Time (h) Fig. 1. Adsorption abilities for nitrate (A) and nitrite (B) of the three kinds of supports.
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with the same SAT of 6 h. For the adsorption of nitrite, the similar adsorption capacities of different carriers were examined as the nitrate adsorption condition. The results indicated that the adsorption abilities of the carriers were closely related to their structures. The relatively weak adsorption (performed by PFCs) may be due to the smaller specific surface areas and fewer pores inside compared with the other two materials. In addition, the powder-activated carbon mixed in the SABs contributed to better performance because of its strong adsorbability (Fatehi et al., 2013). Given the much fewer adsorbance quantity compared with the initial concentration of substrates, adsorption by carriers could be ignored in the following experiments. 3.3. Aerobic denitrification performances of free cells and immobilized cells The aerobic denitrification performances of free cells and different immobilized cells were investigated in DM cultivated at 30 °C and 160 rpm. The same amount of biomass inoculated in the four systems was balanced by the dry weight determination calculation (Table S1). The growth curves of the bacteria represented by OD660 in the four systems were examined, as described in Fig. 2A. Similar trends of growth were observed in the four systems. The bacteria grew rapidly in a period of logarithmic phase during the first 15 h from inoculation. Meanwhile, a slightly higher value of OD660 in MP and PFC systems were observed than the one in the free cells system. However, the maximum OD660 in the PFC system was not as high as the other three systems.
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The better growth in MP and PFC systems maybe due to the better habitats inside the carriers, which could promote the nitrite utilisation by minimising the inhibition of oxygen. In addition, the transfer resistance of substrates in SABs system, caused by the denser structure of material, led to a relatively poor propagation. The nitrate concentrations in the four systems were monitored at an interval of 3 h to estimate their nitrate nitrogen reduction performances, as depicted in Fig. 2B. Excellent nitrate removals were observed in all the four systems. The residual concentrations of NO 3 -N were 7.58, 5.06 and 6.49 mg/L at 18 h in the free cell, MP and PFC systems, with the removal rate of 9.78, 10.20 and 1 1 10.12 mg NO h , respectively. In the SAB system, the 3 -N L NO3 -N concentration in the medium was approximately 20.15 mg/L, along with a slower NO 3 -N removal rate of only 9.36 mg L1 h1. OD660 was proportioned to the value described above (Fig. S3). The accumulation of nitrite and the removal of TN during aerobic denitrification in the four systems were inspected, the results are shown in Fig. 2C for nitrite accumulation and Fig. 2D for TN removal. The TN removal of the free cell system was only 29.7%, with 168.98 mg/L NO 2 -N accumulated in the water. In addition, the higher TN removals of 43.78%, 42.31% and 57.25%, along with NO 2 -N accumulation of 137.4, 142.6 and 89.02 mg/L, were determined in the MP, PFC and SAB systems, respectively. Thus, the TN removal is closely bound with the accumulation of nitrite during aerobic denitrification, as proven in the previous study (Sun et al., 2015). In the present study, the same result occurred in the free cell system and immobilized cell systems. The more nitrite
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Fig. 2. Aerobic denitrification performance of the immobilization systems. (A) Growth curve of bacteria; (B) nitrate depletion; (C) nitrite accumulation; (D) removal of TN.
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3.5. Stress tests of immobilized systems
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DO concentration of the environment (Sun et al., 2015). In this study, another method of immobilization to improve nitrite reduction in aerobic condition was adopted. This approach is usually used in simultaneous nitrification and denitrification (SND) processes. The gradient concentration of dissolved oxygen existed from outer to inner parts of the carriers by limiting mass passing, which could provide an anoxic zone to bacteria inside the carriers (Menoud et al., 1999; Virdis et al., 2011). The OD660 value was not as high as the nitrate supplied condition, as shown in Fig. 3A, and the same consequence was discovered in the previous study (Sun et al., 2015). The cell growth of the three immobilized cell systems was slightly better than the free cell system. A nitrite nitrogen removal of 67.66% was observed in the SAB system, which was the highest among the four systems, 1 1 with a removal rate of 6.41 mg NO h . The nitrite nitrogen 2 -N L removal of 55.18% (free cells system), 62.45% (MPs system) and 59.87% (PFCs system) were also observed, with the removal rate 1 1 of 5.20, 5.94 and 5.69 mg NO h , respectively. 2 -N L The nitrite was mainly reduced by the bacteria suspended in the water, which expressed relatively low activity of nitrite reductase in aerobic condition in the free cell system. However, with the exception of the contribution of suspension cells in the immobilized systems, the immobilized bacteria inside the carriers played an important role to achieve the nitrite reduction. These results verified the feasibility of improving the nitrite removal of strain T13 during aerobic denitrification by immobilization.
50 40 30 20 Free cells Cells immobilized by mycelial pellets Cells immobilized by polyurethane foam cubes Cells immobilized by sodium alginate beads
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Time (h) Fig. 3. Impact of immobilization on the nitrite reduction ability of strain T13. (A) Growth curve of bacteria; (B) nitrite depletion; (C) removal of nitrite.
nitrogen was accumulated, the lower TN removal was achieved. Fortunately, the immobilization resulted in varying improvements for restricting nitrite accumulation. The limiting of the oxygen transfer through immobilization was effective (Uemoto et al., 2000). The bacteria inside the carriers could express the high activities of nitrite reductase, which could be inhibited under a high dissolved oxygen condition (Körner and Zumft, 1989). 3.4. Impact of immobilization on nitrite reduction ability of strain T13 The nitrite reduction process could be inhibited in high dissolved oxygen condition (Ka et al., 1997). The reduction of nitrite by strain T13 could be promoted effectively by decreasing the
The stress test was conducted to investigate the performance on TN removal during aerobic denitrification of the free cell system and the three immobilized cell systems under extreme conditions, such as temperature, pH and shaking speed. 3.5.1. Temperature The TN removal of the four systems under different temperature conditions (10, 20, 25, 30 and 35 °C) is shown in Fig. 4A. Temperature plays a key role in biochemical reaction, and the microbes are often sensitive to temperature variation (Zaitsev et al., 2008). In this study, temperature had a pronounced influence on TN removal by strain T13. The highest TN removal efficiency was achieved at 30 °C in all the four systems. Increasing TN removal percentage of each system was observed with the increase in cultivation temperature from 10 °C to 30 °C. The clearest change of the PFC system was taken as example, the TN removal was only 13.42% at 10 °C, but increased evidently to 54.56% when the temperature was increased to 30 °C. However, TN removal no longer increased when the temperature was further increased to 35 °C. In addition, the TN removal efficiency decreased by 3.69% as the temperature dropped from 25 °C to 20 °C in the MP system, compared with the 6.32% in the free cell system, accounting for the protection of the carriers from decreasing temperature. However, the poorest behaviour of the SAB system at 10 °C was caused by the limited transfer rate combined with the inactivation of microbes. 3.5.2. pH The activities and functions of most microorganisms could be impaired by an overly basic or acidic condition (Xiao et al., 2013; Yu et al., 2014). Without exception, the aerobic denitrification capacity of strain T13 remarkably decreased as the initial pH was adjusted to acidic or alkaline. The optimal pH value for TN removal was 7 in all the four systems, with the highest removal efficiency of 29.82%, 43.78%, 42.31% and 54.56% for the free cell, MP, PFC and SAB systems, respectively (Fig. 4B). The poorest performance of each system was observed when the initial pH was 5, in which
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Fig. 5. Reuse of immobilized cells on aerobic denitrification.
aerobic denitrification process. Under high dissolved oxygen condition (160 rpm), the gap of TN removal between the free cell system and the immobilization systems was realised by the protection of carriers from excess oxygen. However, the advantages of immobilization systems were no longer obvious under low dissolved oxygen condition (50 rpm). On the other hand, the broken SABs occurred at the too high shaking speed of 200 rpm, causing the decline of TN removal efficiency as the same level of free cells. 3.6. Reuse of immobilized cells on aerobic denitrification
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The recycling of immobilized cells was evaluated to inspect their potentials for further practical application. After each cycle, the carriers and the medium were washed three times to clear the free cells for the next cycle. At the end of each cycle, the removal of TN was measured, as shown in Fig. 5. From the first cycle to the seventh cycle, a stable performance was observed in the MP and PFC systems, the removal of TN settled around 41%. Nevertheless, a decreasing trend was observed in the SAB system. In the first cycle, a high TN removal efficiency of 55.56% was observed, followed by a slight decrease to 52.95% in the second cycle. In the third cycle, more loose structure of SABs appeared, the SABs were completely dissolved in the fourth cycle, with a low removal efficiency of 28.34% as the same level of free cells. The reason behind the broken SABs was mainly caused by the high strength phosphate in the medium, which was usually used as the solvent of solid sodium alginate (Muyima and Cloete, 1995).
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the activity of the microbes was affected seriously. Notably, the SABs were dissolved with the initial pH of 9, causing significant decrease in TN removal efficiency. 3.5.3. Shaking speed Adjusting the shaking speed of cultivation could change the reoxygenation rate to provide different dissolved oxygen conditions to microbes, as well as test the mechanical strengths of the carriers. On one hand, the removal of TN in the four systems could be promoted by decreasing the rotation speed from 160 rpm to 50 rpm, in which the removal percentages increases to more than 80% (Fig. 4C).The activity of nitrite reductase could be stimulated to reduce the accumulated nitrite because of the low dissolved oxygen conditions provided to the microorganisms during the
4. Conclusions The aerobic denitrification capabilities of the three immobilization systems (MPs, PFCs and SABs) were investigated. The pathway of accumulated nitrite reduction was activated successfully through inhibition of oxygen inside the carriers. In addition, nitrite reduction as the rate-limiting step during aerobic denitrification process had been broken by immobilization. A maximum TN removal efficiency of 57.25% could be promoted by immobilization (in SABs system), compared with 29.7% by free cells. Moreover, the MP and PFC systems were still stable on TN removal after 7 cycles. Therefore, these results showed the potential advances of aerobic denitrification combined immobilization for further practical application. Acknowledgement This work was supported by Grants from the National Natural Science Foundation of China (51108120, 51478140 and 51178139), the Open Project of State Key Laboratory of Urban
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Water Resource and Environment, Harbin Institute of Technology, China (HC201526-01). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.biortech.2015.03. 060. References APHA, 2012. Standard methods for the examination of water and wastewater, 22nd ed. American Public Health Association, Washington, D.C., USA. Fatehi, P., Ryan, J., Ni, Y., 2013. Adsorption of lignocelluloses of model pre-hydrolysis liquor on activated carbon. Bioresour. Technol. 131, 308–314. Isaka, K., Kimura, Y., Osaka, T., Tsuneda, S., 2012. High-rate denitrification using polyethylene glycol gel carriers entrapping heterotrophic denitrifying bacteria. Water Res. 46 (16), 4941–4948. Ji, B., Wang, H., Yang, K., 2013. Tolerance of an aerobic denitrifier (Pseudomonas stutzeri) to high O2 concentrations. Biotechnol. Lett. 36, 719–722. Ka, J.O., Urbance, J., Ye, R.W., Ahn, T.Y., Tiedje, J.M., 1997. Diversity of oxygen and Noxide regulation of nitrite reductases in denitrifying bacteria. FEMS Microbiol. Lett. 156, 55–60. Körner, H., Zumft, W.G., 1989. Expression of denitrification enzymes in response to the dissolved oxygen level and respiratory substrate in continuous culture of Pseudomonas stutzeri. Appl. Environ. Microbiol. 55, 1670–1676. Li, A., Gai, Z., Cui, D., Ma, F., Yang, J., Zhang, X., Sun, Y., Ren, N., 2012. Genome sequence of a highly efficient aerobic denitrifying bacterium, Pseudomonas stutzeri T13. J. Bacteriol. 194, 5720. Liang, S., Zhao, M., Lu, L., Wang, C., Zhao, L., Liu, W., 2011. Isolation and characteristic of an aerobic denitrifier with high nitrogen removal efficiency. Afr. J. Biotechnol. 10, 10648–10656. Menoud, P., Wong, C.H., Robinson, H.A., Farquhar, A., Barford, J.P., Barton, G.W., 1999. Simultaneous nitrification and denitrification using Siporax™ packing. Water Sci. Technol. 40 (4), 153–160. Muyima, N.Y.O., Cloete, T.E., 1995. Growth and phosphate uptake of immobilized Acinetobacter cells suspended in activated sludge mixed liquor. Water Res. 29 (11), 2461–2466. Nair, I.C., Jayachandran, K., Shashidhar, S., 2007. Treatment of paper factory effluent using a phenol degrading Alcaligenes sp. under free and immobilized conditions. Bioresour. Technol. 98 (3), 714–716. Padhi, S.K., Tripathy, S., Sen, R., Mahapatra, A.S., Mohanty, S., Maiti, N.K., 2013. Characterization of heterotrophic nitrifying and aerobic denitrifying Klebsiella
pneumoniae CF-S9 strain for bioremediation of wastewater. Int. Biodeterior. Biodegradation 78, 67–73. Robertson, L.A., Kuenen, J.G., Kleijntjens, R., 1985. Aerobic denitrification and heterotrophic nitrification by Thiosphaera pantotropha. Antonie Van Leeuwenhoek 51, 445. Scholten, E., Lukow, T., Auling, G., Kroppenstedt, R.M., Rainey, F.A., Diekmann, H., 1999. Thauera mechernichensis sp. nov., an aerobic denitrifier from a leachate treatment plant. Int. J. Syst. Bacteriol. 49, 1045–1051. Shi, S., Qu, Y., Ma, F., Zhou, J., 2014. Bioremediation of coking wastewater containing carbazole, dibenzofuran and dibenzothiphene by immobilized naphthalenecultivated Arthrobacter sp. W1 in magnetic gellan gum. Bioresour. Technol. 166, 79–86. Sparacino-Watkins, C., Stolz, J.F., Basu, P., 2014. Nitrate and periplasmic nitrate reductases. Chem. Soc. Rev. 43, 676–706. Sun, Y., Li, A., Zhang, X., Ma, F., 2015. Regulation of dissolved oxygen from accumulated nitrite during the heterotrophic nitrification and aerobic denitrification of Pseudomonas stutzeri T13. Appl. Microbiol. Biotechnol. 99, 3243–3248 Uemoto, H., Ando, A., Saiki, H., 2000. Effect of oxygen concentration on nitrogen removal by Nitrosomonas europaea and Paracoccus denitrificans immobilized within tubular polymeric gel. J. Biosci. Bioeng. 90 (6), 654–660. Van Suijdam, J.C., Hols, H., Kossen, N.W.F., 1982. Unstructured model for growth of mycelial pellets in submerged cultures. Biotechnol. Bioeng. 24 (1), 177–191. Van Suijdam, J.C., Kossen, N.W.F., Paul, P.G., 1980. An inoculum technique for the production of fungal pellets. Appl. Microbiol. Biotechnol. 10 (3), 211–221. Virdis, B., Read, S.T., Rabaey, K., Rozendal, R.A., Yuan, Z.G., Keller, J., 2011. Biofilm stratification during simultaneous nitrification and denitrification (SND) at a biocathode. Bioresour. Technol. 102 (1), 334–341. Xiao, Y., Zhang, X., Zhu, M., Tan, W., 2013. Effect of the culture media optimization, pH and temperature on the biohydrogen production and the hydrogenase activities by Klebsiella pneumoniae ECU-15. Bioresour. Technol. 137, 9–17. Yu, R., Shi, L., Gu, G., Zhou, D., You, L., Chen, M., Qiu, G., Zeng, W., 2014. The shift of microbial community under the adjustment of initial and processing pH during bioleaching of chalcopyrite concentrate by moderate thermophiles. Bioresour. Technol. 162, 300–307. Zaitsev, G., Mettänen, T., Langwaldt, J., 2008. Removal of ammonium and nitrate from cold inorganic mine water by fixed-bed biofilm reactors. Miner. Eng. 21 (1), 10–15. Zhang, J., Wu, P., Hao, B., Yu, Z., 2011a. Heterotrophic nitrification and aerobic denitrification by the bacterium Pseudomonas stutzeri YZN-001. Bioresour. Technol. 102, 9866–9869. Zhang, S., Li, A., Cui, D., Yang, J., Ma, F., 2011b. Performance of enhanced biological SBR process for aniline treatment by mycelial pellet as biomass carrier. Bioresour. Technol. 102 (6), 4360–4365. Zhu, G., Peng, Y., Li, B., Guo, J., Yang, Q., Wang, S., 2008. Biological removal of nitrogen from wastewater. Rev. Environ. Contam. Toxicol. 192, 159–195.
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Activation of accumulated nitrite reduction by immobilized Pseudomonas stutzeri T13 during aerobic denitrification Fang Ma, Yilu Sun, Ang Li ⇑, Xuening Zhang, Jixian Yang State Key Laboratory of Urban Water Resource and Environment, Harbin Institute of Technology, Harbin 150090, People’s Republic of China
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
Reduction of accumulated nitrite
could be promoted by immobilization. A high total nitrogen (TN) removal could be achieved in immobilization systems. The performance of mycelial pellets and polyurethane foam cubes system were steady.
a r t i c l e
i n f o
Article history: Received 13 February 2015 Received in revised form 10 March 2015 Accepted 11 March 2015 Available online 24 March 2015 Keywords: Aerobic denitrification Accumulation of nitrite TN removal Immobilization Pseudomonas stutzeri T13
a b s t r a c t The excellent removal efficiency of nitrate by the aerobic denitrifier, Pseudomonas stutzeri T13, was achieved in free cells system. However, poor nitrite reduction prevents efficient aerobic denitrification because of the nitrite accumulation. This problem could be conquered by immobilizing the cells on supports. In this study, strain T13 was immobilized by mycelial pellets (MPs), polyurethane foam cubes (PFCs) and sodium alginate beads (SABs). Higher removal percentages of TN in MP (43.78%), PFC (42.31%) and SAB (57.25%) systems were achieved compared with the free cell system (29.7%). Furthermore, the optimal condition for immobilized cell systems was as follows: 30 °C, 100 rpm shaking speed and pH 7. The shock-resistance of SAB system was relatively poor, which could collapse under either alkaline (pH = 9) or high rotating (200 rpm) conditions. The recycling experiments demonstrated that the high steady TN removal rate could be maintained for seven cycles in both MP and PFC systems. Ó 2015 Elsevier Ltd. All rights reserved.
1. Introduction Aerobic denitrification as a novel biological technology of nitrogen removal that could successfully overcome the incompatibility of nitrification with denitrification caused by different oxygen demands has gained a great of attention in recent years (Zhu et al., 2008). Since the first microbe with special function of ⇑ Corresponding author at: State Key Laboratory of Urban Water Resource and Environment, School of Municipal and Environmental Engineering, Harbin Institute of Technology, Harbin 150090, People’s Republic of China. Tel./fax: +86 451 86283787. E-mail address: [email protected] (A. Li). http://dx.doi.org/10.1016/j.biortech.2015.03.060 0960-8524/Ó 2015 Elsevier Ltd. All rights reserved.
reducing nitrate to gaseous nitrogen aerobically has been found (Robertson et al., 1985), some novel aerobic denitrifiers have been isolated and reported, such as Thauera mechernichensis sp. nov. TL1T (Scholten et al., 1999), Pseudmonas stutzeri YZN-001 (Zhang et al., 2011a) and Klebsiella pneumonia CF-S9 (Padhi et al., 2013). Aerobic denitrifiers could conduct an aerobic respiratory process, in which nitrate is gradually reduced to N2 (Ji et al., 2013). The periplasmic nitrate reductase (Nap) located on the periplasmic side of the cell membrane of the aerobic denitrifier could guarantee the slight inhibition of nitrate reduction by dissolved oxygen (DO) (Sparacino-Watkins et al., 2014). However, nitrite reductases (Nir) in aerobic denitrification bacteria are still sensitive to oxygen,
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similar to traditional denitrifiers (Körner and Zumft, 1989). In our previous report, reduction of nitrite has been proven to be the ratelimiting step during aerobic denitrification, especially under high nitrate loading condition, which could be improved by adjusting the DO to a relatively low level (Sun et al., 2015).In addition, accumulation of nitrite as the end-product instead of gaseous nitrogen from the nitrate reduction process would decrease the removal efficiency of total nitrogen (TN) severely (Körner and Zumft, 1989; Liang et al., 2011). Therefore, effective methods to break this bottleneck and lay the foundation for further practical application of aerobic denitrification are necessary. Microbial immobilized technology has been widely used in biological treatment of contaminants (Isaka et al., 2012; Nair et al., 2007; Shi et al., 2014; Zhang et al., 2011b). The limited O2 transfer rate as a defect of immobilized cells could be regarded as an advantage from a certain perspective. The gradient concentration of dissolved oxygen in the inner spaces of the supports would provide an effective area for accumulated nitrite reduction during aerobic denitrification. In this study, three kinds of supports, namely, mycelial pellets (MPs), polyurethane foam cubes (PFCs) and sodium alginate beads (SABs), were used for immobilizing strain T13 to improve the aerobic denitrification TN removal performance. The nitrogen removal efficiency and the accumulation of nitrite in free cell and immobilized cell systems were compared. The results provided an efficient method to activate the reduction of nitrite accumulating during aerobic denitrification and improve TN removal. 2. Methods 2.1. Strains and media P. stutzeri T13 and Aspergillus niger Y3 were provided by the State Key Laboratory of Urban Water Resource and Environment, Harbin Institute of Technology, China. In previous study, strain T13 was confirmed to be an aerobic denitrification bacterium and exhibited an excellent performance on nitrate reduction under aerobic condition (Li et al., 2012; Sun et al., 2015). Strain Y3 was used to flocculate into the mycelial pellet under the submerged shaking condition. The denitrification medium (DM) used for T13 strain cultivation contained (per litter of distilled water) 4.7 g of sodium succinate, 1.5 g of KNO3, 1.5 g of KH2PO4, 7.9 g of Na2HPO4, 0.5 g of MgSO47H2O and 1 ml of a trace element solution, with pH 7.0– 7.2. The trace element solution contained (per litter of distilled water) 50 g of EDTA, 2.2 g of ZnSO4, 5.5 g of CaCl2, 5.06 g of MnCl24H2O, 5.0 g of FeSO47H2O, 1.1 g of (NH4)6Mo7O24H2O, 1.57 g of CuSO45H2O and 1.61 g of CoCl26H2O.The liquid medium (LM) for culturing strain Y3 into mycelial pellet contained (per litter of distilled water) 10 g of glucose, 1.0 g of KH2PO4, 1.0 g of NH4Cl and 0.5 g of MgSO4 (Zhang et al., 2011b). 2.2. Preparation of mycelial pellet and polyurethane foam The spores of strain Y3 on the solid medium were washed into a 100 ml flask containing several glass beads with the distilled water under sterile condition. The spore suspension was obtained after gentle shaking and the absorbance was measured with a visible spectrophotometer under the wavelength of 620 nm to quantify the spore concentration (Van Suijdam et al., 1980; Van Suijdam et al., 1982). The spore suspension of strain Y3 was then inoculated into LM to cultivate the mycelial pellet at 30 °C and 140 rpm. The inoculation quantity was calculated as follows:
Volume of spore suspension for inoculation ¼
0:25 0:1% volume of liquid medium absorbance of spore suspension ðAÞ
31
The polyurethane foam was cut into approximately 0.5 cm cubes, washed three times with distilled water, and then autoclaved (121 °C, 30 min) before used. The average dry weight of MPs and PFCs were measured. 2.3. Preparation of free and immobilized cells Strain T13, grown in DM for 24 h was harvested by centrifugation at 10,000 rpm for 5 min. The cells were suspended in sterile water at a cell concentration of 1 OD660, and the dry weight of 1 ml suspension was determined to quantify the biomass. A certain amount of homogeneous MPs and PFCs were added into two 1 L flasks containing 200 mL of DM, and then inoculated with 10 mL cell suspension. The flasks were incubated at 30 °C on a shaker at 160 rpm for 24 h (one cycle). Afterwards, the supernatant was thrown out and fresh DM was added instead. Immobilization could be finished after 4–5 cycles, as a large number of biomass was adsorbed visibly in the inside and outside the MPs (Fig. S1A) and PFCs (Fig. S1B). The average dry weights of the carriers after immobilization were measured. The quantity of biomass immobilized by either per mycelial pellet or polyurethane foam cube was calculated as follows: The average biomass immobilized by per carrier ðgÞ ¼
dry weight after immobilization ðgÞ dry weight before immobilization ðgÞ number of carriers
The sodium alginate and powder-activated carbon were mixed at the dry weight ratio of 8 (wt/wt) and dissolved in the distilled water to compound the 4% sodium alginate solution. The autoclaved solution and T13 cell suspension at a cell concentration of 1 OD660 were mixed. The mixture was extruded through a syringe into 4% CaCl2 solution and solidified for 24 h at 4 °C to obtain the alginate entrapped cells (Fig. S1C). After immobilization, the cells adsorbed by three different carriers were probed by scanning electron microscopy (SEM). 2.4. Adsorption abilities of nitrate and nitrite in different carriers The same quality (5 g) of the three kinds of carriers (MPs, PFCs and SABs) without cells were added in triplicate into 250 mL flasks each containing 100 mL of DM. A medium without additive was used as control. The nitrate concentration was regularly determined to estimate the adsorption of nitrate nitrogen by the carriers. The adsorption of carriers on nitrite nitrogen was conducted in DM containing nitrite instead of nitrate. All experiments were performed in triplicate. In this part, for accurate measurement, the concentrations of nitrate and nitrite were determined by ion chromatography. 2.5. Aerobic denitrification performance of free and immobilized cells To investigate the effect of different immobilization carriers on the aerobic denitrification performance of strain T13, the free cells (cell suspension of T13) and immobilized cells (MPs, PFCs and SABs) were inoculated into four 250 mL shaking flasks each containing 100 mL of DM. The inoculum biomass was approximately equivalent to the control by calculating the dry weight of the biomass at different conditions (Table S1). A medium without inoculation was used as control. All flasks were covered with gaspermeable seals to provide an aerobic condition to the systems, and then cultivated with constant shaking at 160 rpm and 30 °C for 24 h. The samples were obtained from the flasks at 3-h intervals to determine the optical density at 660 nm (OD660), as well as the concentrations of nitrate nitrogen (NO 3 -N), nitrite nitrogen (NO2 N) and total nitrogen (TN). Each experiment was performed in triplicate.
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2.6. Impact of immobilization on the nitrite reduction ability of strain T13 To evaluate the effect of immobilization on nitrite reduction of strain T13 during aerobic denitrification, the equivalent biomass of the free cells and the three different kinds of immobilized cells were inoculated in four 250 mL flasks with 100 mL of DM containing nitrite instead of nitrate. A non-inoculated medium was used for comparison. The flasks were cultivated aerobically at 160 rpm and 30 °C for 24 h. The samples were regularly collected at 3-h intervals to measure the optical density at 660 nm (OD660), as well as the concentration of nitrite nitrogen (NO 2 -N) and the removal percentage of nitrite was calculated. Each experiment was performed in triplicate.
hand, a large number of biomass was observed in the inner spaces of PFCs. For SABs, the colour of the carriers changed from glossy black to grey during immobilization. Thus, colour changes could be distinguished through macroscopic observation between the carriers with cells and the pure ones. After immobilization, the MPs, PFCs and SABs were analysed by SEM (Fig. S2). A large amount of biomass was accessibly immobilized by all kinds of carriers. Although the internal structure of the polyurethane foam was not as compact as the other two carriers, sufficient biomass was guaranteed by the thick biofilms (Fig. S2B). Moreover, the biomass located inside the carriers were observed more than the biomass on the surface. It demonstrated that the strain T13 tended to grow at relatively low dissolved oxygen circumstance which could be provided inside the carriers through mass transfer limitation.
2.7. Stress tests of the immobilized systems Batch experiments were conducted to examine the effects of temperature, pH and shaking speed on TN removal of the different immobilized systems. In the first set of experiments, the cultivation temperatures were 10, 20, 25, 30 and 35 °C, and the experiments were conducted at initial pH of 7 and shaking speed of 160 rpm. In the second set of experiments, the initial pH value was adjusted to 5, 6, 7, 8 and 9, and the experiment was conducted at 30 °C and shaking speed of 160 rpm. In the third set of experiments, the shaking speed was controlled at 0, 50, 100, 160 and 200 rpm, and the experiment was carried out at initial pH of 7 and 30 °C. In each batch, the sample was obtained at 18 h and the removal of TN was analysed. All the experiments were performed in triplicate.
3.2. Adsorption abilities for nitrate and nitrite by different carriers The adsorption abilities for nitrate and nitrite by different carrier materials were investigated, the results are shown in Fig. 1. Differences in the adsorption abilities on nitrate (Fig. 1A) or nitrite (Fig.1B) between the three carrier materials were observed. For the adsorption of nitrate, SABs performed the best adsorption capacity among the three carriers, with saturated adsorption of 10.71 mg/L. The NO 3 -N in the medium was adsorbed rapidly during the first 3 h and remained constant. Meanwhile, the approximately 3 h of saturated adsorption time (SAT) of SABs was the shortest among the other two materials. By contrast, the specific maximum adsorption of MPs and PFCs for nitrate was 7.28 and 5.32 mg/L
2.8. Reuse of immobilized cells on aerobic denitrification
2.9. Analytical methods of water quality
3. Results and discussion 3.1. Morphological characteristics of the carriers after immobilization In the process of immobilization, for MPs and PFCs, the cells of strain T13 were immobilized by adsorption firstly. Afterwards, the biomass on carriers was mainly enlarged by propagation. Both kinds of carriers after immobilization seemed obviously different from the ones without cells. On one hand, the colour of MPs before immobilization was white, but changed to beige after immobilization, similar to that of the strain T13 colony (Fig. S1A). On the other
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The concentrations of ammonium, nitrate, nitrite and TN were determined according to standard methods, i.e., by Nessler’s reagent spectrophotometry, phenol disulfonic acid photometry, N-(1-naphthyl)-ethylene diamine photometry and alkaline potassium persulfate photometry, respectively (APHA, 2012). The growth of T13 was measured using a spectrophotometer at a wavelength of 660 nm.
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The recycling experiments using the three kinds of immobilized cells to reduce nitrate during aerobic denitrification in three 250 mL flasks containing 100 mL of DM were carried out. The experiments were conducted at initial pH of 7, shaking speed of 100 rpm and 30 °C. After each batch, the immobilization materials were collected in a clean bench and washed three times with sterile water to remove the free cells. Afterwards, the immobilized cells were added separately into the fresh DM for the next cycle. Each cycle lasted for 18 h. At the end of each cycle, the OD660 value and removal of TN were examined.
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with the same SAT of 6 h. For the adsorption of nitrite, the similar adsorption capacities of different carriers were examined as the nitrate adsorption condition. The results indicated that the adsorption abilities of the carriers were closely related to their structures. The relatively weak adsorption (performed by PFCs) may be due to the smaller specific surface areas and fewer pores inside compared with the other two materials. In addition, the powder-activated carbon mixed in the SABs contributed to better performance because of its strong adsorbability (Fatehi et al., 2013). Given the much fewer adsorbance quantity compared with the initial concentration of substrates, adsorption by carriers could be ignored in the following experiments. 3.3. Aerobic denitrification performances of free cells and immobilized cells The aerobic denitrification performances of free cells and different immobilized cells were investigated in DM cultivated at 30 °C and 160 rpm. The same amount of biomass inoculated in the four systems was balanced by the dry weight determination calculation (Table S1). The growth curves of the bacteria represented by OD660 in the four systems were examined, as described in Fig. 2A. Similar trends of growth were observed in the four systems. The bacteria grew rapidly in a period of logarithmic phase during the first 15 h from inoculation. Meanwhile, a slightly higher value of OD660 in MP and PFC systems were observed than the one in the free cells system. However, the maximum OD660 in the PFC system was not as high as the other three systems.
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The better growth in MP and PFC systems maybe due to the better habitats inside the carriers, which could promote the nitrite utilisation by minimising the inhibition of oxygen. In addition, the transfer resistance of substrates in SABs system, caused by the denser structure of material, led to a relatively poor propagation. The nitrate concentrations in the four systems were monitored at an interval of 3 h to estimate their nitrate nitrogen reduction performances, as depicted in Fig. 2B. Excellent nitrate removals were observed in all the four systems. The residual concentrations of NO 3 -N were 7.58, 5.06 and 6.49 mg/L at 18 h in the free cell, MP and PFC systems, with the removal rate of 9.78, 10.20 and 1 1 10.12 mg NO h , respectively. In the SAB system, the 3 -N L NO3 -N concentration in the medium was approximately 20.15 mg/L, along with a slower NO 3 -N removal rate of only 9.36 mg L1 h1. OD660 was proportioned to the value described above (Fig. S3). The accumulation of nitrite and the removal of TN during aerobic denitrification in the four systems were inspected, the results are shown in Fig. 2C for nitrite accumulation and Fig. 2D for TN removal. The TN removal of the free cell system was only 29.7%, with 168.98 mg/L NO 2 -N accumulated in the water. In addition, the higher TN removals of 43.78%, 42.31% and 57.25%, along with NO 2 -N accumulation of 137.4, 142.6 and 89.02 mg/L, were determined in the MP, PFC and SAB systems, respectively. Thus, the TN removal is closely bound with the accumulation of nitrite during aerobic denitrification, as proven in the previous study (Sun et al., 2015). In the present study, the same result occurred in the free cell system and immobilized cell systems. The more nitrite
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DO concentration of the environment (Sun et al., 2015). In this study, another method of immobilization to improve nitrite reduction in aerobic condition was adopted. This approach is usually used in simultaneous nitrification and denitrification (SND) processes. The gradient concentration of dissolved oxygen existed from outer to inner parts of the carriers by limiting mass passing, which could provide an anoxic zone to bacteria inside the carriers (Menoud et al., 1999; Virdis et al., 2011). The OD660 value was not as high as the nitrate supplied condition, as shown in Fig. 3A, and the same consequence was discovered in the previous study (Sun et al., 2015). The cell growth of the three immobilized cell systems was slightly better than the free cell system. A nitrite nitrogen removal of 67.66% was observed in the SAB system, which was the highest among the four systems, 1 1 with a removal rate of 6.41 mg NO h . The nitrite nitrogen 2 -N L removal of 55.18% (free cells system), 62.45% (MPs system) and 59.87% (PFCs system) were also observed, with the removal rate 1 1 of 5.20, 5.94 and 5.69 mg NO h , respectively. 2 -N L The nitrite was mainly reduced by the bacteria suspended in the water, which expressed relatively low activity of nitrite reductase in aerobic condition in the free cell system. However, with the exception of the contribution of suspension cells in the immobilized systems, the immobilized bacteria inside the carriers played an important role to achieve the nitrite reduction. These results verified the feasibility of improving the nitrite removal of strain T13 during aerobic denitrification by immobilization.
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Time (h) Fig. 3. Impact of immobilization on the nitrite reduction ability of strain T13. (A) Growth curve of bacteria; (B) nitrite depletion; (C) removal of nitrite.
nitrogen was accumulated, the lower TN removal was achieved. Fortunately, the immobilization resulted in varying improvements for restricting nitrite accumulation. The limiting of the oxygen transfer through immobilization was effective (Uemoto et al., 2000). The bacteria inside the carriers could express the high activities of nitrite reductase, which could be inhibited under a high dissolved oxygen condition (Körner and Zumft, 1989). 3.4. Impact of immobilization on nitrite reduction ability of strain T13 The nitrite reduction process could be inhibited in high dissolved oxygen condition (Ka et al., 1997). The reduction of nitrite by strain T13 could be promoted effectively by decreasing the
The stress test was conducted to investigate the performance on TN removal during aerobic denitrification of the free cell system and the three immobilized cell systems under extreme conditions, such as temperature, pH and shaking speed. 3.5.1. Temperature The TN removal of the four systems under different temperature conditions (10, 20, 25, 30 and 35 °C) is shown in Fig. 4A. Temperature plays a key role in biochemical reaction, and the microbes are often sensitive to temperature variation (Zaitsev et al., 2008). In this study, temperature had a pronounced influence on TN removal by strain T13. The highest TN removal efficiency was achieved at 30 °C in all the four systems. Increasing TN removal percentage of each system was observed with the increase in cultivation temperature from 10 °C to 30 °C. The clearest change of the PFC system was taken as example, the TN removal was only 13.42% at 10 °C, but increased evidently to 54.56% when the temperature was increased to 30 °C. However, TN removal no longer increased when the temperature was further increased to 35 °C. In addition, the TN removal efficiency decreased by 3.69% as the temperature dropped from 25 °C to 20 °C in the MP system, compared with the 6.32% in the free cell system, accounting for the protection of the carriers from decreasing temperature. However, the poorest behaviour of the SAB system at 10 °C was caused by the limited transfer rate combined with the inactivation of microbes. 3.5.2. pH The activities and functions of most microorganisms could be impaired by an overly basic or acidic condition (Xiao et al., 2013; Yu et al., 2014). Without exception, the aerobic denitrification capacity of strain T13 remarkably decreased as the initial pH was adjusted to acidic or alkaline. The optimal pH value for TN removal was 7 in all the four systems, with the highest removal efficiency of 29.82%, 43.78%, 42.31% and 54.56% for the free cell, MP, PFC and SAB systems, respectively (Fig. 4B). The poorest performance of each system was observed when the initial pH was 5, in which
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Free cells Cells immobilized by mycelial pellets Cells immobilized by polyurethane foam cubes Cells immobilized by sodium alginate beads
Removal of TN (%)
50 40 30 20
5
6
7
8
9
pH
C
100
Free cells Cells immobilized by mycelial pellets Cells immobilized by polyurethane foam cubes Cells immobilized by sodium alginate beads
80
Removal of TN (%)
3
4
5
6
7
Fig. 5. Reuse of immobilized cells on aerobic denitrification.
aerobic denitrification process. Under high dissolved oxygen condition (160 rpm), the gap of TN removal between the free cell system and the immobilization systems was realised by the protection of carriers from excess oxygen. However, the advantages of immobilization systems were no longer obvious under low dissolved oxygen condition (50 rpm). On the other hand, the broken SABs occurred at the too high shaking speed of 200 rpm, causing the decline of TN removal efficiency as the same level of free cells. 3.6. Reuse of immobilized cells on aerobic denitrification
10 0
2
Cycles
o
60
40
20
The recycling of immobilized cells was evaluated to inspect their potentials for further practical application. After each cycle, the carriers and the medium were washed three times to clear the free cells for the next cycle. At the end of each cycle, the removal of TN was measured, as shown in Fig. 5. From the first cycle to the seventh cycle, a stable performance was observed in the MP and PFC systems, the removal of TN settled around 41%. Nevertheless, a decreasing trend was observed in the SAB system. In the first cycle, a high TN removal efficiency of 55.56% was observed, followed by a slight decrease to 52.95% in the second cycle. In the third cycle, more loose structure of SABs appeared, the SABs were completely dissolved in the fourth cycle, with a low removal efficiency of 28.34% as the same level of free cells. The reason behind the broken SABs was mainly caused by the high strength phosphate in the medium, which was usually used as the solvent of solid sodium alginate (Muyima and Cloete, 1995).
0 0
50
100
160
200
Shaking speed (rpm) Fig. 4. Effects of temperature (A), pH (B) and shaking speed (C) on TN removal by the three immobilization systems.
the activity of the microbes was affected seriously. Notably, the SABs were dissolved with the initial pH of 9, causing significant decrease in TN removal efficiency. 3.5.3. Shaking speed Adjusting the shaking speed of cultivation could change the reoxygenation rate to provide different dissolved oxygen conditions to microbes, as well as test the mechanical strengths of the carriers. On one hand, the removal of TN in the four systems could be promoted by decreasing the rotation speed from 160 rpm to 50 rpm, in which the removal percentages increases to more than 80% (Fig. 4C).The activity of nitrite reductase could be stimulated to reduce the accumulated nitrite because of the low dissolved oxygen conditions provided to the microorganisms during the
4. Conclusions The aerobic denitrification capabilities of the three immobilization systems (MPs, PFCs and SABs) were investigated. The pathway of accumulated nitrite reduction was activated successfully through inhibition of oxygen inside the carriers. In addition, nitrite reduction as the rate-limiting step during aerobic denitrification process had been broken by immobilization. A maximum TN removal efficiency of 57.25% could be promoted by immobilization (in SABs system), compared with 29.7% by free cells. Moreover, the MP and PFC systems were still stable on TN removal after 7 cycles. Therefore, these results showed the potential advances of aerobic denitrification combined immobilization for further practical application. Acknowledgement This work was supported by Grants from the National Natural Science Foundation of China (51108120, 51478140 and 51178139), the Open Project of State Key Laboratory of Urban
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