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Comparison of the effects of different salts on aerobic ammonia oxidizers for treating


Chemosphere 81 (2010) 669–673

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Chemosphere
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Technical Note

Comparison of the effects of different salts on aerobic ammonia oxidizers for treating ammonium-rich organic wastewater by free and sodium alginate immobilized biomass system
Jia Yan a,b, Mike Jetten b, Jinlong Rang a, Yongyou Hu a,c,*
a b c

School of Environmental Science and Engineering, South China University of Technology, Guangzhou 510006, People’s Republic of China Department of Microbiology, IWWR, Radboud University Nijmegen, 6500 GL Nijmegen, The Netherlands Ministry of Education Key Laboratory of Pollution Control and Ecological Remediation for Industrial Agglomeration Area, Department of Environmental Science and Engineering, South China University of Technology, Guangzhou 510006, China

a r t i c l e

i n f o

a b s t r a c t
Partial nitri?cation to nitrite by aerobic ammonium oxidizing bacteria (AOB) is an important pre-treatment step for subsequent denitri?cation and anammox. Ammonium-rich wastewater may contain different amounts of organic matter and salts, which can in?uent the growth and activity of AOB signi?cantly. In this study we investigated the in?uence of various salts on the performance of a partial nitri?cation process with free and sodium alginate immobilized biomass. Immobilization of the AOB cells did not have a great effect on the activity of the biomass, and complete inhibition for the immobilized AOB was observed at sulfate, chloride and phosphate concentrations of 500, 1000 and 700 mM, respectively. Free biomass was already inhibited at 300, 500 and 500 mM concentrations of sulfate, chloride and phosphate. Both free and immobilized biomass contained Nitrosomonas europaea/eutropha-like AOB. Compared to free nitrifying biomass, immobilized biomass appeared to be less sensitive to salt stress (maximum 30%). Since no difference in the composition of the AOB was observed between free and immobilized biomass, the protection by the immobilization is the most likely factor explaining the observed differences. ? 2010 Elsevier Ltd. All rights reserved.

Article history: Received 8 September 2009 Received in revised form 12 March 2010 Accepted 15 March 2010 Available online 24 August 2010 Keywords: Nitri?cation Immobilization Sulfate Chloride Phosphate

1. Introduction Several new processes and operational strategies like combined nitri?cation–denitri?cation and partial nitri?cation–anammox have emerged in order to remove high concentrations of nitrogen compound in wastewaters (Jetten et al., 1997; Fux et al., 2002; Aslan et al., 2009). All these processes are based on the feasibility of partial nitri?cation to nitrite by aerobic ammonium oxidizing bacteria (AOB). The partial nitri?cation of ammonia to nitrite not only reduces the oxygen requirements for the process, but less organic matter is needed for denitri?cation. In the case of combination with anammox no organic matter is needed at all because both groups of bacteria use CO2 for assimilation. Many nitrogenous wastewaters, such as urban land?ll leachate, contain high amounts of organic matter and salts (Kjeldsen et al., 2002). Sulfate, phosphate and chloride salts are the most abundant components of leachate. Autotrophic AOB, which convert ammonium to nitrite, have to compete for oxygen with heterotrophic

* Corresponding author at: School of Environmental Science and Engineering, South China University of Technology, Guangzhou 510006, People’s Republic of China. E-mail address: ppyyhu@scut.edu.cn (Y. Hu). 0045-6535/$ - see front matter ? 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.chemosphere.2010.03.025

bacteria in these ammonium-rich organic wastewater treatment processes, and the free nitri?ers can be easily washed out of the reactor even when long retention times are applied. In addition, AOB are strongly in?uenced by a variety of environmental factors and toxic substrates, including dissolved oxygen (DO), salt, organic carbon and so on (Philips et al., 2002; Rene et al., 2008). Immobilization can be an effective method to prevent biomass from washed out, when environmental factor are not favorable or toxic substrate are present (Wijffels and Tramper, 1995; Ho et al., 2008). Although the procedure is often used to immobilize pure cultures, the method can also be applied to mixed bacterial communities (Rostron et al., 2001; Oliveira et al., 2009). The effect of several environmental factors on immobilized biomass have been studied by various research groups (Isaka et al., 2007; Yan and Hu, 2009), however, the possible in?uence of salt on immobilized biomass has so far not received much attention. Because of the different studies used various operating conditions and microbial communities, the reports on the effect of salt on nitri?cation are dif?cult to compare (Campos et al., 2002; Moussa et al., 2006; Tan et al., 2008). Therefore in this study the in?uences of salt on partial nitri?cation with free and sodium alginate (SA) immobilized biomass, which was used to treat ammonium-rich organic wastewater, were

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investigated. At ?rst, the maximum ammonium oxidizing activity of free and SA immobilized biomass was determined, and then the in?uence of phosphate, sulfate and chloride on free biomass and SA immobilized biomass was investigated. Finally, the AOB present in free and SA immobilized beads were identi?ed by using molecular methods. The aim of this work was to determine if immobilization has an advantage in reactor operation for ammonium-rich organic wastewater with large amount of salts. 2. Materials and methods 2.1. Immobilization methods Biomass was obtained from the activated sludge of the aeration tank of an urban wastewater treatment plant (WWTP) in Guangzhou, China, and acclimated with synthetic ammonia and COD feeding for 8 months in two 5 L sequencing batch reactor (SBR) resulting in stable partial nitrifying biomass. The acclimated biomass was washed by distilled water three times before use. One portion of the concentrated biomass (28.8 g volatile suspended solids L?1) was mixed thoroughly with two portion of the support solution. Then the mixture was pumped into crosslink solution to form solid beads, ?nally, the solid beads was ready to use after a reactive period of 12 h to minimize the effect of immobilized process to the biomass (Yan and Hu, 2009). The particle size of SA immobilization beads were 3.7 ± 0.1 mm. 2.2. Experimental setup and procedure The batch experiments were carried out in 12 simultaneous SBRs of 1 L and a volume exchange ratio of about 0.8. Half of the reactors were used for free biomass and the others were used with immobilized biomass. For immobilized biomass system, 200 mL of immobilized beads was added to each reactor. All the reactors were stirred magnetically. A PVC net was installed at the bottom of the reactor (pore size 1 mm) to keep the immobilized beads away from the rotor. For free biomass system, 300 mL of free biomass was added to each reactor and the PVC net was not installed (70 mL concentrated biomass could be obtained from 300 mL free biomass, and 70 mL concentrated biomass mixed with support solution could ?nally obtain 200 mL immobilized beads, it means the same amount of biomass was used in free and immobilized biomass). All reactors were provided with a thermostatic jacket and temperature was maintained at 30 ± 1 °C using thermostatic bath. One pump (Beta/4a 0708, Prominent, Germany) and oxygen valve were necessary to operate each reactor. All reactors were drawn by gravity discharge using an electro-valve. DO was measured with an electrode (550A, YSI, USA) and controlled at about 4 mg L?1 by adjusting the air ?ow rate manually. The pH was measured with an electrode (pH 6, Ecoscan, Singapore) and adjusted to about 7.5 by addition of 1 M H2SO4 or 1 M NaOH solution, respectively. The feeding medium contained (g L?1): 2.305 NH4Cl, 3.60 HaHCO3, 0.30 CaCl2, 0.07 KH2PO4, 0.02 MgSO4, 0.009 FeSO4?7H2O, 0.006

EDTA, 0.26 C6H12O6 and 1.25 mL traces solution (the trace solution was according to Strous et al. (1998)). The experimental time covered three periods. The feeding medium of each reactor was different in each period. A summary of the feeding conditions for each reactor is given in Table 1. The feeding solution was described as above during the entire experiment but with different salt concentrations. 2.3. Activity assays The maximum aerobic ammonium oxidation activity was measured under fully aerobic condition. Biomass were washed 3–5 times with distilled water (20 and 30 mL for free and immobilized biomass, respectively, the protein concentration was about 6 mg mL?1), then was transferred to 250 mL conical ?ask. One hundred millilitre medium with pH 7.5 was added to the conical ?ask. In order to measure aerobic ammonium oxidization activity, ?nal concentration 1.5 mM ammonium was used. The ?asks were incubated at 30 ± 1 °C and were shaken continuously at 150 rpm. Ammonium, nitrite and nitrate were measured over time during 2 h. 2.4. DNA extraction, polymerase chain reaction (PCR) ampli?cation of amoA and phylogeny inference Total genomic DNA was extracted as described previously (Schmid et al., 2007). The preferential polymerase chain reaction (PCR) ampli?cation of bacterial amoA was performed with amoA forward primer (50 -GGGGTTTCTACTGGTGGT-30 ) in combination with amoA reverse primer (50 -CCCCTCKGSAAAGCCTTCTTC-30 ). PCR was performed with a Tgradient cycler (Biometra, Germany). Negative controls (no DNA added) and positive controls (DNA from a ‘Nitrosomonas eutropha’ pure culture) were included in all sets of ampli?cations. The presence and size of ampli?cation products were determined by agar (1%) gel electrophoresis of 5 lL aliquots of the PCR products. Cloning was performed using the pGEM-T easy vector cloning kit (Promega Corporation, USA) according to the supplier’s instructions. Plasmid DNA was extracted using the E.Z.N.A. Plasmid Mini Kit (Omega BIO-TEK, USA). Clones were checked by restriction analysis of plasmid DNA (EcoR1, Fermentas GMBH, Germany). AmoA gene sequences were obtained by sequencing with primer M13F. The ContigXpress program of the Vector NTI Suite 7.0 software package (InforMax) was used to assemble entire 16S rRNA gene sequences. The amoA gene sequences determined in this study have been deposited in GenBank under accession numbers GQ120563 and GQ141713 to GQ141713. 2.5. Analytical methods Ammonium, nitrites and nitrates were measured according to Standard Methods (APHA, 1995). The volume of immobilized bead was measured by draining method, which measured the volume of mixed liquor displaced when immobilized beads were added.

Table 1 Feeding conditions for each reactor at each period. Period Salta Salt concentration (mM) for each reactor Immobilized biomass system 1 A B C
a

Free biomass system 4 250 500 300 5 300 700 400 6 500 1000 500 7 0 0 0 8 50 100 100 9 100 300 200 10 250 500 300 11 300 700 400 12 500 1000 500

2 50 100 100

3 100 300 200

Sulfate Chloride Phosphate

0 0 0

Salts concentration in feeding medium were ignored.

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Fig. 1. Batch test of aerobic ammonia activity of free and immobilized biomass.

2.6. Experiment data For every salt concentration test, the percentage of nitrite in this study was the percentage of produced nitrite concentration to total ammonium concentration in the medium. All the results of this study were the average values (n > 5), the error bar indicated the relative error. For the study of salt tolerance, the average values were concentration in the ef?uent of reactor after the reactor worked stable. 3. Results and discussion 3.1. Aerobic ammonia oxidizing activity of free and immobilized biomass The characteristics of the free and immobilized biomass were compared using batch tests as shown in Fig. 1. In batch experiments, ammonia oxidizing activity of immobilized biomass was about 10% lower than free biomass. This result indicated that either the AOB were partly damaged during the immobilization process or that the activity was limited by internal mass transfer of substrates (Benyahia and Polomarkaki, 2005). No nitrate was detected during the entire experiment, which might indicated that nitrite oxidizing bacteria were not present. 3.2. Effect of salts on partial nitri?cation by free and immobilized biomass 3.2.1. Sulfate and chloride Fig. 2a and b presents the results obtained in the experiments with sodium sulfate and sodium chloride additions. Ammonium conversion and subsequent nitrite accumulation decreased with increasing sulfate and chloride concentrations. As shown in Fig. 2a, about 40% of ammonia oxidizing activity was lost with the free biomass at 100 mM sulfate, while the decrease with immobilized biomass system was only 25% (nitrite concentration in immobilized biomass system was 1.3 times higher than free biomass system). At 500 mM sulfate no ammonia oxidizing activity was observed either in free or immobilized cells. The inhibitory effect of chloride was less pronounced than that of sulfate in this study (Fig. 2b). Only 25% and 10% ammonia oxidizing activity lost at a chloride concentration of 100 mM in free and immobilized system, respectively (nitrite concentration in immobilized biomass system was 1.2 times higher than free biomass system). Ammonium oxidization was completely inhibited when chloride concentration reached 500 mM with the free cells, while for the

Fig. 2. Effect of sulfate (a), chloride (b) and phosphate (c) on partial nitri?cation by free and immobilized biomass.

immobilized cells a chloride concentration of 1000 mM was necessary to completely inhibit activity. The comparable effects of sulfate and chloride on AOB in the present experiments may be attributed to an increase of osmotic pressure as reported by Hunik et al. (1992). Our inhibition percentages observed at 100 mM are in agreement with previous reports (Hunik et al., 1992; Campos et al., 2002; Mosquera-Corral et al., 2005). Also the effect of chloride in those studies was less severe than that of sulfate additions. The values where complete inhibitions were observed previously are in good agreement with our values namely 525 (Campos et al., 2002), 513 (Mosquera-Corral et al., 2005) and 1127 mM (Moussa et al., 2006), respectively. This result indicated that some halotolerant AOB might be present in

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Fig. 3. Phylogenetic trees (neighbor joining method) of bacterial amoA gene sequences. The bar indicates 5% estimated sequence divergence.

the biomass (see below). The effects of salts on nitrite formation were less pronounced when the cells were immobilized especially in the chloride tests. Besides, it seems that maybe because of the higher charge, the effect of sulfate was higher than chloride. 3.2.2. Phosphate Fig. 2c showed that the effect of phosphate on nitrite formation was similar to those of sulfate, and that at 500 mM activity was completely inhibited for free cells. Appropriate levels of phosphate are necessary for nitri?er growth, the half-saturation coef?cient for AOB is 0.03 mg P L?1 (Nowak et al., 1996). It has been reported that nitri?cation was inhibited by phosphate limitation (van Droogenbroeck and Laudelout, 1967). van Droogenbroeck and Laudelout (1967) also found that the speci?c growth rate of Nitrosomonas was not very sensitive to phosphate below 100 mM which is comparable to our results. 3.3. Microbial population composition In order to get more insight in the microbial community we determined the diversity of amoA genes in both free and immobilized biomass (Fig. 3). DNA was extracted and ampli?ed with amoA speci?c primers. Seven clones of the immobilized and seven clones

of the free cells were analysed and phylogenetic analysis revealed that all the amoA sequences obtained belonged to the Nitrosomonas europaea–eutropha-like cluster. All the amoA sequences were 98– 99% similar to environmental sequences previously recovered from animal wastewater treatment plant (Otawa et al., 2006), leachate treatment sites (Zhu et al., 2007) and an anammox reactor (Quan et al., 2008). Interestingly, all the three wastewater systems mentioned above received in?uent containing high amounts of ammonium, organic compounds and salts. Physiological characteristics such as the maximum ammonia tolerance and maximum salt tolerance have been studied for several AOB strains (Otawa et al., 2006), but it remains dif?cult to predict what kind of AOB will become dominant in certain types of wastewater. In previous studies, AOBs belonging to either N. europaea or N. ureae–oligotropha–marina cluster were major constituents of AOB community in WWTP (Purkhold et al., 2000; Bollmann and Laanbroek, 2001). Furthermore N. europaea was shown to be halotolerant or moderately halophilic ammonia oxidizer. Moussa et al. (2006) reported that both N. oligotropha and N. europaea were present and active when salt was absent, but that only N. europaea were present and active when salt concentrations were above 30 g L?1. Whang et al. (2009) reported that N. europaea-like AOB were the major nitrifying population found in the

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swine waste treating plants. These results are congruent with our ?ndings that the higher salt tolerance in this study may be related to presence of N. europaea-like AOB. No differences in the AOB composition of AOB were observed between free and immobilized biomass. On one hand, this indicated that the immobilization process was not very harmful to the growth and activity of the N. europaea-like AOB, which is consistent with the presence and activity of nitrifying biomass in polyethylene glycol immobilized system for treating land?ll leachate (Isaka et al., 2007), and with high nitri?cation rates in calcium alginate beads that were used in a system fed with sludge digester supernatant (Hill and Khan, 2008). On the other hand, this result also suggested the less severe effect of salt on immobilized biomass was not because of a difference in community composition. 4. Conclusions Compared to free AOB, the immobilized biomass was less sensitive to salt stress. Immobilization could thus be an excellent method for protecting biomass when short-term salt stress may be expected. Completely nitri?cation inhibitions were observed when the concentration of sulfate, chloride and phosphate were 500, 1000 and 700 mM for the immobilized AOB biomass, respectively, while for the free cells 300, 500 and 500 mM were observed. High salt tolerance could be due to the presence of N. europaea/eutrophalike AOB. Acknowledgements Suzanne Haaijer and Erwin van de Biezen of the Department of Microbiology, Radboud University Nijmegen are gratefully acknowledged for technical assistance. This research was supported by NSFC (Project No. 50678071), GSFC (Project No. 06105409) and State Scholarship Fund of China Scholarship Council (2008615074). MJ was supported by the ERC Advanced Grant 232937. References
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