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Nitrogen removal performance of a hybrid anammox reactor
Bioresource Technology 102 (2011) 6650–6656
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Nitrogen removal performance of a hybrid anammox reactor Yongguang Ma ⇑, Daisuke Hira, Zhigang Li, Cheng Chen, Kenji Furukawa Graduate School of Science and Technology, Kumamoto University, 2-39-1 Kurokami, Kumamoto 860-8555, Japan
a r t i c l e
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Article history: Received 23 January 2011 Received in revised form 23 March 2011 Accepted 24 March 2011 Available online 29 March 2011 Keywords: Nitrogen removal Anammox Hybrid reactor Mechanical stirrer Granular characteristics
a b s t r a c t The anaerobic ammonium oxidation (anammox) process has attracted considerable attention in recent years as an alternative to conventional nitrogen removal technologies. In this study, an innovative hybrid reactor combining fluidized and fixed beds for anammox treatment was developed. The fluidized bed was mechanically stirred and the gaseous product could be rapidly released from the anammox sludge to prevent washout of the sludge caused by floatation. The fixed bed comprising a non-woven biomass carrier could efficiently catch sludge to reduce washout. During the operation, nitrogen loading rates to the reactor were increased to 27.3 kg N/m3/d, with total nitrogen removal efficiencies of 75%. The biomass concentration in the fluidized bed reached 26-g VSS/L. Anammox granules were observed in the reactors, with settling velocities and sludge volumetric index of 27.3 ± 6.5 m/h and 23 mL/g, respectively. Quantification of extracellular polymeric substances revealed the anammox granules contained a significant amount of extracellular proteins. Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction One of major issues in wastewater treatment, nitrogen removal, has attracted increasing attention due to the potential for nitrogen to cause eutrophication. The nitrification–denitrification process has been widely used for nitrogen removal in wastewater treatment. However, a large amount of oxygen and exogenous carbon sources are required for nitrification and denitrification, respectively. Anaerobic ammonium oxidation (anammox) is a novel and promising process for the removal of inorganic nitrogen from wastewater treatment (Mulder et al., 1995; Van de Graaf et al., 1995). In the anammox reaction, ammonium (NHþ 4 AN) is aerobically utilized as the electron donor for the reduction of nitrite (NO 2 AN), yielding di-nitrogen gas (N2) as the final product.
NHþ4 þ 1:32NO2 þ 0:066HCO3 þ 0:13Hþ ! 1:02N2 þ 0:26NO3 þ 2:03H2 O þ 0:066CH2 0:5N0:15
ð1Þ
A partial nitritation–anammox process is potentially a costeffective alternative to the conventional nitrification–denitrification process. In this process, only half of the ammonium has to be oxidized partly to nitrite and then anammox bacteria oxidize ammonium to nitrogen gas using nitrite as an electron accepter under anoxic conditions, with their growth occurring by carbon dioxide fixation. Compared to the conventional nitrification–denitrification-dependent nitrogen removal systems, the partial nitrita⇑ Corresponding author. Tel./fax: +81 96 342 3544. E-mail address: [email protected] (Y. Ma). 0960-8524/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2011.03.081
tion–anammox requires less oxygen and no exogenous carbon source (e.g., methanol). Anammox bacteria have extremely low growth rates (a doubling time of 11 days) and the biomass yield of the anammox process is very low so only a small amount of excess sludge is produced during treatment. The low sludge production is the third factor that contributes to the substantially lower operation costs compared to conventional denitrification systems. However, due to the low biomass yield of anammox bacteria, reactors that can effectively retain biomass and provide a long solids retention time are desirable for successful and efficient operation of the anammox process. Biofilm type reactors, such as fixed bed, fluidized bed and gas lift reactors have initially been applied for anammox treatment (Van de Graaf et al., 1996; Strous et al., 1997; Sliekers et al., 2003). The sequencing batch reactor was also applied to anammox sludge cultivation (Strous et al., 1997, 1999; Dapena-Mora et al., 2004). Fixed-bed reactors using various types of biomass carriers, e.g., polyethylene (PE) sponge (Zhang et al., 2010), polyester non-woven material (Furukawa et al., 2001), novel acrylic resin material (Qiao et al., 2009), poly (vinyl alcohol)-gel beads (Hoa et al., 2006) and polyethylene glycol gel (Isaka et al., 2007), were developed for stable immobilization of anammox sludge. The development of new types of anammox reactor and their application to actual wastewater treatment has been a challenging problem. Among anaerobic wastewater treatment technologies, hybrid reactors combining the advantages of both sludge blanket and fixed-bed reactors in having both matrix-free and support matrix regions have been developed. The support matrix region retains the suspended sludge within the reactor and treats the wastewater through the activity of the biofilm developed on the packing mate-
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rial. Although the hybrid reactor design has been successfully applied to the treatment of several wastewaters (Colleran et al., 1994; Ozturk et al., 1993; Borja et al., 1995), there are no studies of its application to the anammox process. In this study, a hybrid reactor design with two parts, combining fluidized and fixed beds, was developed. The fluidized bed was mechanically stirred to increase the mixing efficiency between wastewater and anammox sludge and to release the gaseous products from the sludge. The fixed bed using a non-woven biomass carrier was designed to effectively catch the suspended sludge and reduce its washout. Rapid startup of the anammox system, anammox granular enrichment and the high nitrogen removal efficiency of this hybrid anammox reactor were investigated experimentally.
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analyzed by the colorimetric method (APHA/AWWA/WPCF, 1995). DO and pH were determined using a DO meter (D-55, Horiba, Japan) and a pH meter (B-211, Horiba, Japan), respectively. Particle size analysis was conducted by a laser scattering particle size distribution analyzer (LA-920, Horiba, Japan). MLSS was measured by drying at 105 °C on an evaporating dish. Granule settling velocities were measured by timing the settling time of individual granules taken from the bottom of the reactor. Extracellular polymeric substances (EPS) were extracted from sludge by formaldehyde plus NaOH (Liu and Fang, 2002) and the proteins were measured using the method of Lowry et al. (1951) and carbohydrates by the method of Dubois et al. (1956). 2.4. SEM observation
2. Methods 2.1. Experimental setup and operational strategy The hybrid anammox reactor was made of acrylic resin and had an effective volume of 6.0 L, an internal diameter of 120 mm and a height/diameter ratio of 4.2. The reactor comprised a fluidized bed in the lower part (0–250 mm from the bottom) and a fixed bed in the upper part (250–500 mm from the bottom). A mechanical stirrer (Z-2200, Tokyo rikakikai, Japan) was installed from the top of the reactor to the lower part. A flat paddle (diameter 90 mm) and a turbine stirrer (diameter 60 mm) with six blades made of a stainless steel were used. The flat paddle and the turbine stirrers were located at ca. 50 and 200 mm from the bottom, respectively, to mechanically fluidize the lower part (Fig. 1). In the fluidized bed, no carrier particles were used for granule formation. The upper part was filled with a porous polyester non-woven fabric carrier (Japan Vilene, Tokyo, Japan). Ten bundles of the carrier were inserted in the upper part with an apparent volume of 1.25 L (21% of the total reactor volume). Two gas–liquid–solid separators (GSS) were attached to the reactor, located 260 mm (lower port) and 520 mm (higher port) from the bottom (Fig. 1). The reactor was enclosed by a black vinyl sheet to inhibit the growth of photosynthetic bacteria. The temperature was controlled at around 33–37 °C by a water jacket. The influent pH was kept at around 7.0–7.4. The influent storage tank was flushed with nitrogen gas to maintain an influent dissolved oxygen (DO) concentration below 1 mg/L in the reactors. The reactor was operated in up-flow mode with the influent introduced from the bottom section by a peristaltic pump (RP-2000, Tokyo rikakikai, Japan). 2.2. Seed anammox sludge and feeding media
The surface and inner parts of the anammox granules were observed using a scanning electron microscope (SEM). Samples were first washed in a 0.1 M phosphate buffer solution (pH 7.4) for 5 min. The samples were then hardened for 90 min in a 2.5% glutaraldehyde solution prepared with the 0.1 M phosphate buffer solution (pH 7.4). Next, samples were washed in the buffer solution three times for 10 min each and fixed for 90 min in a 1.0% OsO4 solution prepared using 0.1 M phosphate buffer solution (pH 7.4). After washing samples three times for 10 min each in the buffer solution, they were dewatered for 10 min each in serially graded solutions of ethanol at concentrations of 10%, 30%, 50%, 70%, 90% and 95%. SEM observations were conducted using a scanning electron microscope (JEOL, JSM-5310LV, Japan). 2.5. DNA extraction and PCR amplification A sludge sample was taken from the hybrid reactor at day 250. The granular sludge sample was first ground with a pestle under liquid nitrogen. Meta-genomic DNA was extracted using an ISOIL kit (Wako, Osaka, Japan) according to the manufacturer’s instructions. The amplification of 16S rRNA gene was performed with Phusion High-Fidelity DNA polymerase (FINNZYMES Finland) using conserved eubacterial primers 6F (forward primer: 50 -GGAGA GTTAGATCTTGGCTCAG-30 ) (Tchelet et al., 1999) and 1492r (reverse primer: 50 -GGTTACCTTGTTACGACT-30 ) (Lane, 1991). PCR was carried out according to the following thermocycling parameters: 30 s initial denaturation at 98 °C, 25 cycles of 10 s at 98 °C, 20 s at 51 °C, 35 s at 72 °C, and 5 min final elongation at 72 °C. The amplified products were electrophoresed on a 1% agarose gel and the excised fragments were purified using a Wizard SV Gel and PCR Clean-Up System (Promega, USA).
Seed anammox sludge containing KSU-1 and KU2 strains (Furukawa et al., 2001; Fujii et al., 2002) was collected from an anammox reactor operated in our laboratory. The enriched anammox granular sludge (accounting for 80% of the mixed liquor suspended solids (MLSS)) was taken from a 22 L up-flow fixed-bed reactor using a polyethylene (PE) non-woven fabric as biomass carrier and fed with synthetic wastewater (Furukawa et al., 2003). The reactor was seeded with 24.8 g MLSS of anammox sludge, giving an initial concentration of about 4-g MLSS/L. In this study, the reactor was fed with synthetic wastewater þ with an NO 2 AN to NH4 AN molar ratio of 1.0–1.2. During the operational period, synthetic wastewater with compositions of NHþ 4 AN 50–300 mg/L, NO 50–300 mg/L, KHCO3 125–500 mg/L, 2 AN KH2PO4 54 mg/L, FeSO47H2O 9 mg/L and EDTA 5 mg/L was used.
2.6. Cloning and sequencing of 16S rRNA gene
2.3. Analytical method
3.1. The treatment performance of the anammox reactor
NHþ 4 AN was measured by a modified phonate method using ortho-phenyl phenol (Kanda, 1995). NO 2 AN and NO3 AN were
The reactor performance was monitored for 335 days. The treatment results for continuous operation of the reactor are shown in
The purified fragments were ligated into the EcoRV site of pBluescript II KS+ (Stratagene, USA) and E. coli DH 5a was transformed using the constructed plasmids. White colonies including the insert were randomly chosen and the plasmids were extracted by the alkaline method. The nucleotide sequences were determined with a 3130xl genetic analyzer and BigDye terminator v3.1 cycle sequencing kit (Applied Biosystems, USA). The sequences determined in this study were compared with the sequences in the nrdatabase using the basic local alignment search tool (BLAST) program available on the NCBI website. 3. Results and discussion
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Fig. 1. Schematic of the hybrid anammox reactor.
Fig. 2. During the 335 days of operation, the influent total nitrogen (TN) concentration was progressively increased from 100 to 600 mg/L and the hydraulic retention time (HRT) was decreased from 8 to 0.4 h with an average nitrite removal efficiency of 85% and an average total nitrogen removal efficiency of 75%. A nitrogen loading rate (NLR) of 27.3 kg N/m3/d and corresponding nitrogen removal rate (NRR) of 20.7 kg N/m3/d were obtained after 335 days of operation. 3.1.1. Start-up period The anammox reactor was started up with a TN concentration of 100 mg/L and an HRT of 8 h. During days 1–22, intermittent stirring (run 5 min, stop 30 min) was initially applied and the NLR was about 0.35 kg N/m3/d but total nitrogen removal efficiency was only around 50%. During days 22–39 continuous stirring (30 rpm) and internal circulation were applied and the NLR was increased rapidly from 0.35 to 1.2 kg N/m3/d by increasing the influent TN concentration and decreasing the HRT. During this period, the effluent NO 2 AN concentration remained below 10 mg/L, with a TN removal efficiency of 80 ± 2%. This shows that continuous stirring and internal circulation are important during the start-up period.
Compared with previous studies of the anammox reactor, this hybrid reactor exhibited a relatively high adaptability to the rapid increase in NLR (Liu et al., 2008; Zhang et al., 2010; Yang et al., 2010). Liu et al. (2008) reported that the NLR reached 2.1 kg N/ m3/d on day 50, with an initial anammox volatile suspended solids (VSS) concentration of 0.8-g VSS/L in a non-woven biological rotating contactor. Zhang et al. (2010) reported that the NLR was increased to 1.3 kg N/m3/d on day 56, with an inoculated 4-g MLSS/L of anammox biomass in an up-flow column reactor. Yang et al. (2010) reported that the NLR was increased to 2.5 kg N/m3/ d on day 24, with an initial anammox sludge concentration of 4g MLSS/L in an up-flow column reactor having a spiral-style GSS. In addition to a high concentration of initial anammox sludge (about 4-g MLSS/L), it was suggested that the operational strategy of applying continuous stirring (30 rpm) and internal circulation was useful for rapid start-up of the hybrid reactor. 3.1.2. NLR increasing During days 39–197, the hybrid reactor was operated under continuous stirring (30–100 rpm) with no internal circulation, to increase the NLR. By step-wise increases in influent ðNO 2 AN and NHþ 4 AN concentrations and reduction in the HRT, the NLR was
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Fig. 2. Time courses of total NLR, NRR, nitrogen removal efficiency and influent and effluent nitrogen concentrations, along with HRT.
increased from 1.2 to 8.7 kg N/m3/d. The influent TN concentration was increased from 100 to 600 mg/L and the HRT decreased from 3.0 to 1.5 h. A maximum NRR of 6.3 kg N/m3/d was obtained during this period, with a nitrogen removal efficiency of 75%. After the TN concentration reached 600 mg/L, during days 197–251, the TN concentration was maintained at around 550 mg/L and the HRT was decreased from 1.5 to 0.9 h. As a result of this operation, a maximum NLR of 16.0 kg N/m3/d was obtained, with a TN removal efficiency above 80%. 3.1.3. Recovery from operational problems On day 252, the electric power supply was lost so influent could not be introduced and the temperature could not be controlled for about 24 h. The temperature therefore decreased to 18 °C and the NRR was dramatically decreased. During days 253–261, the NLR was decreased to 3.7 kg N/m3/d by increasing the HRT to 2.7 h. þ By step-wise increase in influent NO 2 AN and NH4 AN concentra-
tions and reduction of the HRT, the NRR was increased by day 307 to the same level as that before the power loss. This result indicates that the hybrid anammox reactor can easily recover from operational problems. On day 335, the sludge concentration in the reactor reached 26g VSS/L and a low effluent SS (<30 mg/L) was obtained. The HRT was shortened to 0.4 h and the highest NLR of 27.3 kg N/m3/d, with total nitrogen removal efficiency above 75%, was achieved. The maximum NRR of 20.7 kg N/m3/d was obtained on day 333. There have been only a few studies reported to date with an NRR above 20 kg N/m3/d for the anammox process (Tsushima et al., 2007; Tang et al., 2010, 2011). Tsushima et al. (2007) reported that total NRR was increased to 26.0 kg N/m3/d using up-flow fixedbed glass biofilm column reactors having a liquid volume of 0.8 L. Tang et al. (2010, 2011) reported record high total NRRs of 45.2 and 76.7 kg N/m3/d with HRTs of 0.22 and 0.16 h, respectively. In their studies, up-flow anaerobic sludge blanket reactors with a
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Fig. 3. Changes in granule size distribution and mean diameter during operation.
Table 1 Diameter, settling velocity, sludge volumetric index (SVI5) and EPS contents of the seed sludge and the granules in the reactor at the end of the experiment. Seed sludge
Hybrid reactor
Diameter (lm) Settling velocity (m/h) SVI5 (ml/g-MLVSS)
200 N.D 58
730 27 23
EPS (mg/g-MLVSS) Proteins Polysaccharides PN/PS
47 31 1.5
91 28 3.3
N.D.: not determined.
working volume of 1.1 L were used and it was shown that a high flow rate at low substrate concentrations was more effective in achieving a high NRR than the application of high substrate concentrations at low flow rates. In our study, the NLR was also increased, mainly due to a decreased HRT. Additionally, our hybrid reactor had an effective volume of 6.0 L, which is larger than that of earlier reported reactors having NRRs above 20 kg N/m3/d. It was shown that this hybrid reactor design is applicable to the anammox process, giving high NLRs and NRRs for wastewater treatment. 3.2. Sludge characteristics Granular sludge was successfully cultivated in this study. The sludge was characterized by mean granular diameter, settling velocity, sludge volumetric index (SVI5), EPS quantification and SEM observation. The granule diameter was measured from day 25 to day 349. Fig. 3 shows the change in the mean granule diameter and the diameter distribution. During days 29–150, the mean diameter increased gradually from 250 to 730 lm and thereafter, the mean diameter was maintained at around 700 lm. The percentage of granules with a diameter smaller than 1 mm ranged between 75% and 85%. The granules attained a high settling property over the course of this study. At the end of the experiment, the settling velocity and the SVI5 were measured (Table 1). The settling velocity was 27.3 ± 6.5 m/h. The SVI5 decreased from 58-mL/g MLVSS in the seed sludge to 23-mL/g MLVSS. Yang et al. (2011) reported that the SVI30 decreased to 8-mL/g MLVSS when 3% NaCl was supplied in synthetic wastewater. Tang et al. (2011) also reported a high settling velocities of 73–88 m/h and SVI5s of 24–25-mL/g MLVSS. By comparison with these values, the anammox granule produced in the hybrid reactor can be considered to have good settling perfor-
mance. The formation of granules with good settling properties resulted in high anammox sludge concentrations in the hybrid reactor in spite of operation with stirring (100 rpm) and relatively short HRTs. EPSs are supposed to assist in the formation of microbial granules, regardless of whether the biomass is in suspended or biofilm states. EPSs are considered to be a rich matrix of polymers, mainly including polysaccharides and proteins. In this study, the EPS concentrations were determined at different operational periods. The proteins and the polysaccharide concentrations were 91 ± 12 and 28 ± 8-mg/g MLVSS, respectively, at the end of the experiment (Table 1). The proteins to polysaccharides ratio (PN/PS) is usually used to evaluate the granular settleability and strength (Quarmby and Forster, 1995; Batstone and Keller, 2001; Franco et al., 2006), where a higher PN/PS ratio of granules indicates lower strength and weaker settleability (Quarmby and Forster, 1995; Batstone and Keller, 2001). The low PN/PS ratio of the anammox granules suggests an excellent granular stability. On the other hand, Wu et al. (2009) reported that the secretion of extracellular protein by heterotrophic anaerobic granules was stimulated under high hydrodynamic shear forces in an internal circulation anaerobic reactor. Tang et al. (2011) also reported that the secretion of extracellular protein was enhanced at a higher rate than that of polysaccharides, leading to an increased PN/PS ratio of 2.29 when the HRT was short, i.e., the hydrodynamic shear force was increased. In this study, the PN/PS ratio at an NLR of 27 kg N/m3/d had a relatively high value of 3.3, which would probably be due to the high hydrodynamic shear forces in the hybrid reactor. Although this over-production of extracellular proteins might have the potential to induce sludge floatation, the formation of granules with good settling would result in the accumulation of high concentrations of sludge in this study. The morphological characteristics of anammox biomass were observed using SEM. Sludge samples were collected at the end of the experiment. The micro-organization structure exhibited a higher degree of sphericity and smoothness (data not shown). This structure would have been formed by the high hydrodynamic shear forces caused by the stirring and relatively short HRTs. At the end of operation, the total amount of the biomass on the non-woven fabric carrier in the upper part reached 17-g VSS. The fixed bed was considered to successfully catch the biomass. Considering an effective volume of the upper part of 3.0 L, the biomass concentration in the fixed bed was calculated as 5.7-g VSS/L which was more than four times smaller than the value in the fluidized bed. Therefore, from the viewpoint of the biomass concentration,
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Y. Ma et al. / Bioresource Technology 102 (2011) 6650–6656 Table 2 Homology search results for 16S rRNA gene sequences of the bacterial species in the hybrid reactor. OTU
Taxon
1 2
Planctomycete KSU-1 Uncultured bacterium Uncultured bacterium Uncultured bacterium Uncultured bacterium
3
clone B70 clone FL0428B_PF55 clone: A2 clone: A
Accession
Identity
Number of clones
AB057453 HQ640561 FJ716474 AB462403 AB194898
99–100 98 96 98 97
35 2 1
Operational taxonomic units(OTU) were defined by a 1% distance level in nucleotide sequences.
more than 80% of the total nitrogen removal was thought to be due to the granular sludge in the fluidized bed. These sludge characteristics clearly indicate that the hybrid reactor is useful for cultivation of granular anammox sludge possessing good settling properties. Future studies will focus on the quantification of the effect of the high hydrodynamic shear forces generated by the stirring on the granulation of anammox sludge. In considering the application to real wastewaters, it appears that the systems to directly treat wastewaters without adding external source of nitrite are particularly suitable. The combination of partial nitritation–anammox process has been carried out in a single reactor such as Completely Autotrophic Nitrogen removal Over Nitrite (CANON) and Oxygen-Limited Autotrophic Nitrification–Denitrification (OLAND). The application of this hybrid reactor to CANON process will be explored in future studies. 3.3. DNA analysis The seed sludge used in the hybrid reactor was inoculated from an anammox reactor that contained anammox bacteria strains KSU-1 and KU2 (Furukawa et al., 2001; Fujii et al., 2002). The bacterial community after 250 days of cultivation with synthetic wastewater was analyzed by 16S rRNA gene cloning using universal eubacterial primers. Table 2 shows the results of a homology search for 16S rRNA gene sequences in the community existing in the hybrid reactor. Thirty-five clones had 99–100% sequence identities with the anammox bacterium strain KSU-1 (AB057453). The KSU-1 strain was detected in various anammox reactors seeded with anammox sludge that were cultivated in our laboratory. It was shown that KSU-1 strain was dominant in the hybrid reactor after 250 days. Additionally, two clones had 98% sequence identities with the uncultured bacterium clone B70 (HQ640561) and one with the uncultured bacterium clone: A2 (AB462403), which have been detected in anammox reactors. These results support the conclusion that the hybrid anammox reactor is useful for the enrichment of anammox bacteria. 4. Conclusions A hybrid anammox reactor has been successfully applied to nitrogen removal from synthetic wastewater with relatively high NLR and short HRT. The reactor was operated for 335 days, with a maximum NRR of 20.7 kg N/m3/d and an average total nitrogen removal efficiency of 75%. The anammox granule sludge formed by this operation had a good settling performance and a relatively high PN/PS ratio of 3.3. These results clearly indicate that this hybrid anammox reactor design is useful for achieving a high nitrogen removal rate and the mass cultivation of granular anammox sludge. Acknowledgements This work was supported by Grant-in-Aid for Scientific Research (B) 21310055 (to K.F.) from the Ministry of Education, Cul-
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Van de Graaf, A.A., De Bruijn, P., Robertson, L.A., Jetten, M.S.M., Kuenen, J.G., 1996. Autotrophic growth of anaerobic ammonium-oxidezing microofganisms in a fluidized bed reactor. Appl. Environ. Microbiol. 142, 2187–2196. Wu, J., Zhou, H.M., Li, H.Z., Zhang, P.C., Jiang, J., 2009. Impacts of hydrodynamic shear force on nucleation of flocculent sludge in anaerobic reactor. Water Res. 43, 3029–3036. Yang, J., Zhang, L., Fukuzaki, Y., Hira, D., Furukawa, K., 2010. High-rate nitrogen removal by the Anammox process with a sufficient inorganic carbon source. Bioresour. Technol. 101, 9471–9478. Yang, J., Zhang, L., Hira, D., Fukuzaki, Y., Furukawa, K., 2011. Anammox treatment of high-salinity wastewater at ambient temperature. Bioresour. Technol. 102, 2367–2372. Zhang, L., Yang, J., Ma, Y., Li, Z., Fujii, T., Zhang, W., Nishiyama, T., Furukawa, K., 2010. Treatment capability of an up-flow anammox column reactor using polyethylene sponge strips as biomass carrier. J. Biosci. Bioeng. 110 (1), 72–78.
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Nitrogen removal performance of a hybrid anammox reactor Yongguang Ma ⇑, Daisuke Hira, Zhigang Li, Cheng Chen, Kenji Furukawa Graduate School of Science and Technology, Kumamoto University, 2-39-1 Kurokami, Kumamoto 860-8555, Japan
a r t i c l e
i n f o
Article history: Received 23 January 2011 Received in revised form 23 March 2011 Accepted 24 March 2011 Available online 29 March 2011 Keywords: Nitrogen removal Anammox Hybrid reactor Mechanical stirrer Granular characteristics
a b s t r a c t The anaerobic ammonium oxidation (anammox) process has attracted considerable attention in recent years as an alternative to conventional nitrogen removal technologies. In this study, an innovative hybrid reactor combining fluidized and fixed beds for anammox treatment was developed. The fluidized bed was mechanically stirred and the gaseous product could be rapidly released from the anammox sludge to prevent washout of the sludge caused by floatation. The fixed bed comprising a non-woven biomass carrier could efficiently catch sludge to reduce washout. During the operation, nitrogen loading rates to the reactor were increased to 27.3 kg N/m3/d, with total nitrogen removal efficiencies of 75%. The biomass concentration in the fluidized bed reached 26-g VSS/L. Anammox granules were observed in the reactors, with settling velocities and sludge volumetric index of 27.3 ± 6.5 m/h and 23 mL/g, respectively. Quantification of extracellular polymeric substances revealed the anammox granules contained a significant amount of extracellular proteins. Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction One of major issues in wastewater treatment, nitrogen removal, has attracted increasing attention due to the potential for nitrogen to cause eutrophication. The nitrification–denitrification process has been widely used for nitrogen removal in wastewater treatment. However, a large amount of oxygen and exogenous carbon sources are required for nitrification and denitrification, respectively. Anaerobic ammonium oxidation (anammox) is a novel and promising process for the removal of inorganic nitrogen from wastewater treatment (Mulder et al., 1995; Van de Graaf et al., 1995). In the anammox reaction, ammonium (NHþ 4 AN) is aerobically utilized as the electron donor for the reduction of nitrite (NO 2 AN), yielding di-nitrogen gas (N2) as the final product.
NHþ4 þ 1:32NO2 þ 0:066HCO3 þ 0:13Hþ ! 1:02N2 þ 0:26NO3 þ 2:03H2 O þ 0:066CH2 0:5N0:15
ð1Þ
A partial nitritation–anammox process is potentially a costeffective alternative to the conventional nitrification–denitrification process. In this process, only half of the ammonium has to be oxidized partly to nitrite and then anammox bacteria oxidize ammonium to nitrogen gas using nitrite as an electron accepter under anoxic conditions, with their growth occurring by carbon dioxide fixation. Compared to the conventional nitrification–denitrification-dependent nitrogen removal systems, the partial nitrita⇑ Corresponding author. Tel./fax: +81 96 342 3544. E-mail address: [email protected] (Y. Ma). 0960-8524/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2011.03.081
tion–anammox requires less oxygen and no exogenous carbon source (e.g., methanol). Anammox bacteria have extremely low growth rates (a doubling time of 11 days) and the biomass yield of the anammox process is very low so only a small amount of excess sludge is produced during treatment. The low sludge production is the third factor that contributes to the substantially lower operation costs compared to conventional denitrification systems. However, due to the low biomass yield of anammox bacteria, reactors that can effectively retain biomass and provide a long solids retention time are desirable for successful and efficient operation of the anammox process. Biofilm type reactors, such as fixed bed, fluidized bed and gas lift reactors have initially been applied for anammox treatment (Van de Graaf et al., 1996; Strous et al., 1997; Sliekers et al., 2003). The sequencing batch reactor was also applied to anammox sludge cultivation (Strous et al., 1997, 1999; Dapena-Mora et al., 2004). Fixed-bed reactors using various types of biomass carriers, e.g., polyethylene (PE) sponge (Zhang et al., 2010), polyester non-woven material (Furukawa et al., 2001), novel acrylic resin material (Qiao et al., 2009), poly (vinyl alcohol)-gel beads (Hoa et al., 2006) and polyethylene glycol gel (Isaka et al., 2007), were developed for stable immobilization of anammox sludge. The development of new types of anammox reactor and their application to actual wastewater treatment has been a challenging problem. Among anaerobic wastewater treatment technologies, hybrid reactors combining the advantages of both sludge blanket and fixed-bed reactors in having both matrix-free and support matrix regions have been developed. The support matrix region retains the suspended sludge within the reactor and treats the wastewater through the activity of the biofilm developed on the packing mate-
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rial. Although the hybrid reactor design has been successfully applied to the treatment of several wastewaters (Colleran et al., 1994; Ozturk et al., 1993; Borja et al., 1995), there are no studies of its application to the anammox process. In this study, a hybrid reactor design with two parts, combining fluidized and fixed beds, was developed. The fluidized bed was mechanically stirred to increase the mixing efficiency between wastewater and anammox sludge and to release the gaseous products from the sludge. The fixed bed using a non-woven biomass carrier was designed to effectively catch the suspended sludge and reduce its washout. Rapid startup of the anammox system, anammox granular enrichment and the high nitrogen removal efficiency of this hybrid anammox reactor were investigated experimentally.
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analyzed by the colorimetric method (APHA/AWWA/WPCF, 1995). DO and pH were determined using a DO meter (D-55, Horiba, Japan) and a pH meter (B-211, Horiba, Japan), respectively. Particle size analysis was conducted by a laser scattering particle size distribution analyzer (LA-920, Horiba, Japan). MLSS was measured by drying at 105 °C on an evaporating dish. Granule settling velocities were measured by timing the settling time of individual granules taken from the bottom of the reactor. Extracellular polymeric substances (EPS) were extracted from sludge by formaldehyde plus NaOH (Liu and Fang, 2002) and the proteins were measured using the method of Lowry et al. (1951) and carbohydrates by the method of Dubois et al. (1956). 2.4. SEM observation
2. Methods 2.1. Experimental setup and operational strategy The hybrid anammox reactor was made of acrylic resin and had an effective volume of 6.0 L, an internal diameter of 120 mm and a height/diameter ratio of 4.2. The reactor comprised a fluidized bed in the lower part (0–250 mm from the bottom) and a fixed bed in the upper part (250–500 mm from the bottom). A mechanical stirrer (Z-2200, Tokyo rikakikai, Japan) was installed from the top of the reactor to the lower part. A flat paddle (diameter 90 mm) and a turbine stirrer (diameter 60 mm) with six blades made of a stainless steel were used. The flat paddle and the turbine stirrers were located at ca. 50 and 200 mm from the bottom, respectively, to mechanically fluidize the lower part (Fig. 1). In the fluidized bed, no carrier particles were used for granule formation. The upper part was filled with a porous polyester non-woven fabric carrier (Japan Vilene, Tokyo, Japan). Ten bundles of the carrier were inserted in the upper part with an apparent volume of 1.25 L (21% of the total reactor volume). Two gas–liquid–solid separators (GSS) were attached to the reactor, located 260 mm (lower port) and 520 mm (higher port) from the bottom (Fig. 1). The reactor was enclosed by a black vinyl sheet to inhibit the growth of photosynthetic bacteria. The temperature was controlled at around 33–37 °C by a water jacket. The influent pH was kept at around 7.0–7.4. The influent storage tank was flushed with nitrogen gas to maintain an influent dissolved oxygen (DO) concentration below 1 mg/L in the reactors. The reactor was operated in up-flow mode with the influent introduced from the bottom section by a peristaltic pump (RP-2000, Tokyo rikakikai, Japan). 2.2. Seed anammox sludge and feeding media
The surface and inner parts of the anammox granules were observed using a scanning electron microscope (SEM). Samples were first washed in a 0.1 M phosphate buffer solution (pH 7.4) for 5 min. The samples were then hardened for 90 min in a 2.5% glutaraldehyde solution prepared with the 0.1 M phosphate buffer solution (pH 7.4). Next, samples were washed in the buffer solution three times for 10 min each and fixed for 90 min in a 1.0% OsO4 solution prepared using 0.1 M phosphate buffer solution (pH 7.4). After washing samples three times for 10 min each in the buffer solution, they were dewatered for 10 min each in serially graded solutions of ethanol at concentrations of 10%, 30%, 50%, 70%, 90% and 95%. SEM observations were conducted using a scanning electron microscope (JEOL, JSM-5310LV, Japan). 2.5. DNA extraction and PCR amplification A sludge sample was taken from the hybrid reactor at day 250. The granular sludge sample was first ground with a pestle under liquid nitrogen. Meta-genomic DNA was extracted using an ISOIL kit (Wako, Osaka, Japan) according to the manufacturer’s instructions. The amplification of 16S rRNA gene was performed with Phusion High-Fidelity DNA polymerase (FINNZYMES Finland) using conserved eubacterial primers 6F (forward primer: 50 -GGAGA GTTAGATCTTGGCTCAG-30 ) (Tchelet et al., 1999) and 1492r (reverse primer: 50 -GGTTACCTTGTTACGACT-30 ) (Lane, 1991). PCR was carried out according to the following thermocycling parameters: 30 s initial denaturation at 98 °C, 25 cycles of 10 s at 98 °C, 20 s at 51 °C, 35 s at 72 °C, and 5 min final elongation at 72 °C. The amplified products were electrophoresed on a 1% agarose gel and the excised fragments were purified using a Wizard SV Gel and PCR Clean-Up System (Promega, USA).
Seed anammox sludge containing KSU-1 and KU2 strains (Furukawa et al., 2001; Fujii et al., 2002) was collected from an anammox reactor operated in our laboratory. The enriched anammox granular sludge (accounting for 80% of the mixed liquor suspended solids (MLSS)) was taken from a 22 L up-flow fixed-bed reactor using a polyethylene (PE) non-woven fabric as biomass carrier and fed with synthetic wastewater (Furukawa et al., 2003). The reactor was seeded with 24.8 g MLSS of anammox sludge, giving an initial concentration of about 4-g MLSS/L. In this study, the reactor was fed with synthetic wastewater þ with an NO 2 AN to NH4 AN molar ratio of 1.0–1.2. During the operational period, synthetic wastewater with compositions of NHþ 4 AN 50–300 mg/L, NO 50–300 mg/L, KHCO3 125–500 mg/L, 2 AN KH2PO4 54 mg/L, FeSO47H2O 9 mg/L and EDTA 5 mg/L was used.
2.6. Cloning and sequencing of 16S rRNA gene
2.3. Analytical method
3.1. The treatment performance of the anammox reactor
NHþ 4 AN was measured by a modified phonate method using ortho-phenyl phenol (Kanda, 1995). NO 2 AN and NO3 AN were
The reactor performance was monitored for 335 days. The treatment results for continuous operation of the reactor are shown in
The purified fragments were ligated into the EcoRV site of pBluescript II KS+ (Stratagene, USA) and E. coli DH 5a was transformed using the constructed plasmids. White colonies including the insert were randomly chosen and the plasmids were extracted by the alkaline method. The nucleotide sequences were determined with a 3130xl genetic analyzer and BigDye terminator v3.1 cycle sequencing kit (Applied Biosystems, USA). The sequences determined in this study were compared with the sequences in the nrdatabase using the basic local alignment search tool (BLAST) program available on the NCBI website. 3. Results and discussion
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Fig. 1. Schematic of the hybrid anammox reactor.
Fig. 2. During the 335 days of operation, the influent total nitrogen (TN) concentration was progressively increased from 100 to 600 mg/L and the hydraulic retention time (HRT) was decreased from 8 to 0.4 h with an average nitrite removal efficiency of 85% and an average total nitrogen removal efficiency of 75%. A nitrogen loading rate (NLR) of 27.3 kg N/m3/d and corresponding nitrogen removal rate (NRR) of 20.7 kg N/m3/d were obtained after 335 days of operation. 3.1.1. Start-up period The anammox reactor was started up with a TN concentration of 100 mg/L and an HRT of 8 h. During days 1–22, intermittent stirring (run 5 min, stop 30 min) was initially applied and the NLR was about 0.35 kg N/m3/d but total nitrogen removal efficiency was only around 50%. During days 22–39 continuous stirring (30 rpm) and internal circulation were applied and the NLR was increased rapidly from 0.35 to 1.2 kg N/m3/d by increasing the influent TN concentration and decreasing the HRT. During this period, the effluent NO 2 AN concentration remained below 10 mg/L, with a TN removal efficiency of 80 ± 2%. This shows that continuous stirring and internal circulation are important during the start-up period.
Compared with previous studies of the anammox reactor, this hybrid reactor exhibited a relatively high adaptability to the rapid increase in NLR (Liu et al., 2008; Zhang et al., 2010; Yang et al., 2010). Liu et al. (2008) reported that the NLR reached 2.1 kg N/ m3/d on day 50, with an initial anammox volatile suspended solids (VSS) concentration of 0.8-g VSS/L in a non-woven biological rotating contactor. Zhang et al. (2010) reported that the NLR was increased to 1.3 kg N/m3/d on day 56, with an inoculated 4-g MLSS/L of anammox biomass in an up-flow column reactor. Yang et al. (2010) reported that the NLR was increased to 2.5 kg N/m3/ d on day 24, with an initial anammox sludge concentration of 4g MLSS/L in an up-flow column reactor having a spiral-style GSS. In addition to a high concentration of initial anammox sludge (about 4-g MLSS/L), it was suggested that the operational strategy of applying continuous stirring (30 rpm) and internal circulation was useful for rapid start-up of the hybrid reactor. 3.1.2. NLR increasing During days 39–197, the hybrid reactor was operated under continuous stirring (30–100 rpm) with no internal circulation, to increase the NLR. By step-wise increases in influent ðNO 2 AN and NHþ 4 AN concentrations and reduction in the HRT, the NLR was
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Fig. 2. Time courses of total NLR, NRR, nitrogen removal efficiency and influent and effluent nitrogen concentrations, along with HRT.
increased from 1.2 to 8.7 kg N/m3/d. The influent TN concentration was increased from 100 to 600 mg/L and the HRT decreased from 3.0 to 1.5 h. A maximum NRR of 6.3 kg N/m3/d was obtained during this period, with a nitrogen removal efficiency of 75%. After the TN concentration reached 600 mg/L, during days 197–251, the TN concentration was maintained at around 550 mg/L and the HRT was decreased from 1.5 to 0.9 h. As a result of this operation, a maximum NLR of 16.0 kg N/m3/d was obtained, with a TN removal efficiency above 80%. 3.1.3. Recovery from operational problems On day 252, the electric power supply was lost so influent could not be introduced and the temperature could not be controlled for about 24 h. The temperature therefore decreased to 18 °C and the NRR was dramatically decreased. During days 253–261, the NLR was decreased to 3.7 kg N/m3/d by increasing the HRT to 2.7 h. þ By step-wise increase in influent NO 2 AN and NH4 AN concentra-
tions and reduction of the HRT, the NRR was increased by day 307 to the same level as that before the power loss. This result indicates that the hybrid anammox reactor can easily recover from operational problems. On day 335, the sludge concentration in the reactor reached 26g VSS/L and a low effluent SS (<30 mg/L) was obtained. The HRT was shortened to 0.4 h and the highest NLR of 27.3 kg N/m3/d, with total nitrogen removal efficiency above 75%, was achieved. The maximum NRR of 20.7 kg N/m3/d was obtained on day 333. There have been only a few studies reported to date with an NRR above 20 kg N/m3/d for the anammox process (Tsushima et al., 2007; Tang et al., 2010, 2011). Tsushima et al. (2007) reported that total NRR was increased to 26.0 kg N/m3/d using up-flow fixedbed glass biofilm column reactors having a liquid volume of 0.8 L. Tang et al. (2010, 2011) reported record high total NRRs of 45.2 and 76.7 kg N/m3/d with HRTs of 0.22 and 0.16 h, respectively. In their studies, up-flow anaerobic sludge blanket reactors with a
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Fig. 3. Changes in granule size distribution and mean diameter during operation.
Table 1 Diameter, settling velocity, sludge volumetric index (SVI5) and EPS contents of the seed sludge and the granules in the reactor at the end of the experiment. Seed sludge
Hybrid reactor
Diameter (lm) Settling velocity (m/h) SVI5 (ml/g-MLVSS)
200 N.D 58
730 27 23
EPS (mg/g-MLVSS) Proteins Polysaccharides PN/PS
47 31 1.5
91 28 3.3
N.D.: not determined.
working volume of 1.1 L were used and it was shown that a high flow rate at low substrate concentrations was more effective in achieving a high NRR than the application of high substrate concentrations at low flow rates. In our study, the NLR was also increased, mainly due to a decreased HRT. Additionally, our hybrid reactor had an effective volume of 6.0 L, which is larger than that of earlier reported reactors having NRRs above 20 kg N/m3/d. It was shown that this hybrid reactor design is applicable to the anammox process, giving high NLRs and NRRs for wastewater treatment. 3.2. Sludge characteristics Granular sludge was successfully cultivated in this study. The sludge was characterized by mean granular diameter, settling velocity, sludge volumetric index (SVI5), EPS quantification and SEM observation. The granule diameter was measured from day 25 to day 349. Fig. 3 shows the change in the mean granule diameter and the diameter distribution. During days 29–150, the mean diameter increased gradually from 250 to 730 lm and thereafter, the mean diameter was maintained at around 700 lm. The percentage of granules with a diameter smaller than 1 mm ranged between 75% and 85%. The granules attained a high settling property over the course of this study. At the end of the experiment, the settling velocity and the SVI5 were measured (Table 1). The settling velocity was 27.3 ± 6.5 m/h. The SVI5 decreased from 58-mL/g MLVSS in the seed sludge to 23-mL/g MLVSS. Yang et al. (2011) reported that the SVI30 decreased to 8-mL/g MLVSS when 3% NaCl was supplied in synthetic wastewater. Tang et al. (2011) also reported a high settling velocities of 73–88 m/h and SVI5s of 24–25-mL/g MLVSS. By comparison with these values, the anammox granule produced in the hybrid reactor can be considered to have good settling perfor-
mance. The formation of granules with good settling properties resulted in high anammox sludge concentrations in the hybrid reactor in spite of operation with stirring (100 rpm) and relatively short HRTs. EPSs are supposed to assist in the formation of microbial granules, regardless of whether the biomass is in suspended or biofilm states. EPSs are considered to be a rich matrix of polymers, mainly including polysaccharides and proteins. In this study, the EPS concentrations were determined at different operational periods. The proteins and the polysaccharide concentrations were 91 ± 12 and 28 ± 8-mg/g MLVSS, respectively, at the end of the experiment (Table 1). The proteins to polysaccharides ratio (PN/PS) is usually used to evaluate the granular settleability and strength (Quarmby and Forster, 1995; Batstone and Keller, 2001; Franco et al., 2006), where a higher PN/PS ratio of granules indicates lower strength and weaker settleability (Quarmby and Forster, 1995; Batstone and Keller, 2001). The low PN/PS ratio of the anammox granules suggests an excellent granular stability. On the other hand, Wu et al. (2009) reported that the secretion of extracellular protein by heterotrophic anaerobic granules was stimulated under high hydrodynamic shear forces in an internal circulation anaerobic reactor. Tang et al. (2011) also reported that the secretion of extracellular protein was enhanced at a higher rate than that of polysaccharides, leading to an increased PN/PS ratio of 2.29 when the HRT was short, i.e., the hydrodynamic shear force was increased. In this study, the PN/PS ratio at an NLR of 27 kg N/m3/d had a relatively high value of 3.3, which would probably be due to the high hydrodynamic shear forces in the hybrid reactor. Although this over-production of extracellular proteins might have the potential to induce sludge floatation, the formation of granules with good settling would result in the accumulation of high concentrations of sludge in this study. The morphological characteristics of anammox biomass were observed using SEM. Sludge samples were collected at the end of the experiment. The micro-organization structure exhibited a higher degree of sphericity and smoothness (data not shown). This structure would have been formed by the high hydrodynamic shear forces caused by the stirring and relatively short HRTs. At the end of operation, the total amount of the biomass on the non-woven fabric carrier in the upper part reached 17-g VSS. The fixed bed was considered to successfully catch the biomass. Considering an effective volume of the upper part of 3.0 L, the biomass concentration in the fixed bed was calculated as 5.7-g VSS/L which was more than four times smaller than the value in the fluidized bed. Therefore, from the viewpoint of the biomass concentration,
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Y. Ma et al. / Bioresource Technology 102 (2011) 6650–6656 Table 2 Homology search results for 16S rRNA gene sequences of the bacterial species in the hybrid reactor. OTU
Taxon
1 2
Planctomycete KSU-1 Uncultured bacterium Uncultured bacterium Uncultured bacterium Uncultured bacterium
3
clone B70 clone FL0428B_PF55 clone: A2 clone: A
Accession
Identity
Number of clones
AB057453 HQ640561 FJ716474 AB462403 AB194898
99–100 98 96 98 97
35 2 1
Operational taxonomic units(OTU) were defined by a 1% distance level in nucleotide sequences.
more than 80% of the total nitrogen removal was thought to be due to the granular sludge in the fluidized bed. These sludge characteristics clearly indicate that the hybrid reactor is useful for cultivation of granular anammox sludge possessing good settling properties. Future studies will focus on the quantification of the effect of the high hydrodynamic shear forces generated by the stirring on the granulation of anammox sludge. In considering the application to real wastewaters, it appears that the systems to directly treat wastewaters without adding external source of nitrite are particularly suitable. The combination of partial nitritation–anammox process has been carried out in a single reactor such as Completely Autotrophic Nitrogen removal Over Nitrite (CANON) and Oxygen-Limited Autotrophic Nitrification–Denitrification (OLAND). The application of this hybrid reactor to CANON process will be explored in future studies. 3.3. DNA analysis The seed sludge used in the hybrid reactor was inoculated from an anammox reactor that contained anammox bacteria strains KSU-1 and KU2 (Furukawa et al., 2001; Fujii et al., 2002). The bacterial community after 250 days of cultivation with synthetic wastewater was analyzed by 16S rRNA gene cloning using universal eubacterial primers. Table 2 shows the results of a homology search for 16S rRNA gene sequences in the community existing in the hybrid reactor. Thirty-five clones had 99–100% sequence identities with the anammox bacterium strain KSU-1 (AB057453). The KSU-1 strain was detected in various anammox reactors seeded with anammox sludge that were cultivated in our laboratory. It was shown that KSU-1 strain was dominant in the hybrid reactor after 250 days. Additionally, two clones had 98% sequence identities with the uncultured bacterium clone B70 (HQ640561) and one with the uncultured bacterium clone: A2 (AB462403), which have been detected in anammox reactors. These results support the conclusion that the hybrid anammox reactor is useful for the enrichment of anammox bacteria. 4. Conclusions A hybrid anammox reactor has been successfully applied to nitrogen removal from synthetic wastewater with relatively high NLR and short HRT. The reactor was operated for 335 days, with a maximum NRR of 20.7 kg N/m3/d and an average total nitrogen removal efficiency of 75%. The anammox granule sludge formed by this operation had a good settling performance and a relatively high PN/PS ratio of 3.3. These results clearly indicate that this hybrid anammox reactor design is useful for achieving a high nitrogen removal rate and the mass cultivation of granular anammox sludge. Acknowledgements This work was supported by Grant-in-Aid for Scientific Research (B) 21310055 (to K.F.) from the Ministry of Education, Cul-
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