Start-up of the anammox process from the conventional activated sludge in a hybrid bioreactor

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Journal of Environmental Sciences 2012, 24(6) 1083–1090

JOURNAL OF ENVIRONMENTAL SCIENCES ISSN 1001-0742 CN 11-2629/X

www.jesc.ac.cn

Start-up of the anammox process from the conventional activated sludge in a hybrid bioreactor Xiumei Duan, Jiti Zhou, Sen Qiao ∗, Xin Yin, Tian Tian, Fangdi Xu Key Laboratory of Industrial Ecology and Environmental Engineering, MOE, School of Environmental Science and Technology, Dalian University of Technology, Dalian 116024, China. E-mail: [email protected] Received 13 July 2011; revised 15 August 2011; accepted 10 October 2011

Abstract The anaerobic ammonium oxidation (anammox) process was successfully started up from conventional activated sludge using a hybrid bioreactor within 2 months. The average removal efficiencies of ammonia and nitrite were both over 80%, and the maximum total nitrogen removal rate of 1.85 kg N/(m3 ·day) was obtained on day 362 with the initial sludge concentration of 0.7 g mixed liquor suspended solids (MLSS)/L. Scanning electron microscope (SEM) observation of the granular sludge in the hybrid reactor clearly showed a high degree of compactness and cell sphericity, and the cell size was quite uniform. Transmission electron microscope photos showed that cells were round or oval, the cellular diameter was 0.6–1.0 μm, and the percentage of the anammoxosome compartment was 51%–85% of the whole cell volume. Fluorescence in situ hybridization analysis (FISH) indicated that anammox bacteria became the dominant population in the community (accounting for more than 51% of total bacteria on day 250). Seven planctomycete 16S rRNA gene sequences were present in the 16S rRNA gene clone library generated from the biomass and affiliated to Candidatus Kuenenia stuttgartiensis and Candidatus Brocadia sp., a new anammox species. In addition, the average effluent suspended solid (MLSS) concentrations of outlets I (above the non-woven carrier) and II (below the non-woven carrier) were 0.0009 and 0.0035 g/L, respectively. This showed that the non-woven carrier could catch the biomass effectively, which increased biomass and improved the nitrogen removal rate in the reactor. Key words: anammox; hybrid bioreactor; fluorescence in situ hybridization; 16S rRNA gene clone library DOI: 10.1016/S1001-0742(11)60871-1

Introduction The anammox process can directly oxidize ammonium under anaerobic conditions, with nitrite as the electron acceptor, to produce nitrogen gas without addition of external organic sources. It is a novel and promising alternative to the conventional nitrogen removal processes because of the significant reduction of aeration costs and exogenous electron donor saving, efficient nitrogen removal capacity (Tang et al., 2011), low sludge production and less emission of greenhouse gases (Tsushima et al., 2007; Kampschreur et al., 2008). Especially, the anammox process shows the potential to treat nonbiodegradable organic substrates or low C/N ratio wastewater, such as sludge digester effluent, landfill leachate and farming waste. However, the anammox cell grows slowly, and the doubling time is about 11 days (Strous et al., 1998). To catch the suspended sludge and reduce sludge washout, and promptly achieve high-rate nitrogen removal in an anammox reactor, researchers have adopted many kinds of bioreactors. Hu et al. (2006) achieved steady state with ammonia and nitrite removal percentages higher than 95% * Corresponding author. E-mail: [email protected]

after an operation of 72 days adopting a sequencing batch reactor. Zheng et al. (2004) adopted an upflow anaerobic sludge blanket reactor and started up successfully with nitrifying granular sludge, and the volumetric total nitrogen loading rate (NLR) and total nitrogen removal rate (NRR) were 1.41 and 1.39 kg N/(m3 ·day) respectively. Trigo et al. (2006) used a submerged hollow fiber membrane sequencing batch reactor to retain the biomass and NRR reached up to 0.71 kg N/(m3 ·day) with almost full nitrite removal. Tsushima et al. (2007) inoculated activated sludge containing more anammox cells into an up flow fixed bed biofilm column reactor with non-woven fabric sheets as biomass carrier, and the anammox reaction occurred within 50 days and a total NRR of 26.0 kg N/(m3 ·day) was achieved after 247 days cultivation. Ma et al. (2011) directly seeded anammox sludge (accounting for 80% of the mixed liquor suspended solids) into an combining fluidized and fixed beds hybrid reactor; 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, and the fixed bed was filled with a non-woven biomass carrier to efficiently catch sludge. During the operation, the NLR of the reactor increased to 27.3 kg N/(m3 ·day),

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with total nitrogen removal efficiency of 75%. The biomass concentration in the fluidized bed reached 26 g mixed liquor volatile suspended solids (MLVSS)/L. In the present research, we adopted a similar hybrid reactor with that of Ma et al. (2011) to investigate the startup and performance of anammox sludge cultivation from conventional activated sludge, and investigated the role of the non-woven carrier on the nitrogen removal. We also analyzed bacterial community shifts in detail. Electron microscope observation was applied to investigate bacterial morphology, and fluorescence in situ hybridization (FISH) analysis and 16S rRNA clone library tests were applied to investigate the bacterial community structure.

1 Materials and methods 1.1 Experimental set-up The experimental system is shown in Fig. 1. The hybrid reactor was made of a polymethyl methacrylate column with internal diameter of 80 mm and height of 480 mm. The total volume and working volume were 2.8 and 2 L, respectively. The reactor comprised a fluidized bed in the lower part (0–240 mm from the bottom) and a fixed bed in the upper part (240–440 mm from the bottom). The upper part was filled with a non-woven fabric carrier. Six bundles of the carrier were inserted in the upper part with a volume of 1.0 L (50% of the total reactor volume). Two outlets (I and II), located 440 mm (higher port) and 240 mm (lower port) from the bottom, were analyzed to estimate the effect of the non-woven carriers on anammox reaction. Inoculated sludge was obtained from Lingshui Sewage Treatment Plant, Dalian, China. The seeding sludge had a final mixed liquor suspended solid (MLSS) concentration of 0.7 g/L in the reactor. The hybrid reactor was operated at a recirculation to inflow ratio of 1:1 (V/V) to reduce inhibition to the anammox sludge caused by toxic nitrite. The reactor was enclosed by a black plastic sheet. The Gas vent Outlet I Three phase separator Recirculation pump

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temperature was controlled at 35–37°C with a thermostatic water jacket. The influent pH level was adjusted 7.5–7.8, but not controlled in the reactor. 1.2 Reactor operation and wastewater The anammox reactor was operated for about 1 year, which was divided into two phases. In phase 1 (day 1–220) anammox enrichment and performance of the anammox reactor was tested. In phase 2 (day 220–362) the hydraulic retention time (HRT) was shortened, and the volumetric nitrogen loading rate and volumetric nitrogen removal rate were observed. In the phase (day 315–362), the nitrogen concentrations and the granule floatation of outlets I and II were monitored to estimate the role of the non-woven carrier. The hybrid reactor was fed with synthetic wastewater containing (g/L): KH2 PO4 (0.054), FeSO4 ·7H2 O (0.009), MgSO4 ·7H2 O (0.001), EDTA-2Na (0.005), KCl (0.0014), CaCl2 ·2H2 O (0.0014), NaCl (0.001) and KHCO3 (0.125). Trace nutrient solutions I and II were added at 1 mL/L each. The mineral medium was composed as described by Van de Graaf et al. (1996). NH4 + -N and NO2 − -N were supplied in the form of (NH4 )2 SO4 and NaNO2 , respectively. 1.3 Specific anammox activity assays Anaerobic batch experiments were carried out in a serum bottle of total volume 120 mL containing 100 mL medium. Biomass was harvested from the reactor described above. The biomass sample was washed three times with the mineral medium to remove the residual nitrogen from the reactor, and centrifuged at 6000 r/min for 12 min, the supernatant was drained. The 2.0 g concentrated weight biomass (0.0264 g-MLVSS) was then transferred to the serum bottles above. The pH was adjusted to 7.5 and the temperature was maintained at (35 ± 1)°C. The liquid was purged with nitrogen gas for 10 min to remove O2 . Initial ammonium and nitrite concentrations were 50 mg N/L each. The maximum specific anammox activity was estimated from the peak of the curve plotting the change of the ratios of ammonium and nitrite concentrations to biomass concentration in the vials with the passage of time. 1.4 Analytical methods

Outlet II Hybrid reactor Thermostat

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The influent and effluent samples were collected every three days and were analyzed immediately. The concentrations of nitrite and nitrate were determined using ion-exchange chromatography (ICS-1100, Dionex, USA) with an IonPac AS18 anion column after filtration with 0.22 μm pore size membranes. NH4 + -N, SS, MLSS and MLVSS concentrations were measured according to the standard methods (APHA, 1995), the average particle diameters were analyzed with a Malvern Laser Particle Sizer Analyzer (Mastersizer 2000, Malvern, England). 1.5 Sludge analysis

Fig. 1 Schematic diagram of anammox system representing the flow pattern of the anammox process.

1.5.1 Probe design and FISH FISH assay was carried out to confirm the anammox bacteria and total populations in the reactor using a fluo-

Start-up of the anammox process from the conventional activated sludge in a hybrid bioreactor

1.5.2 Electron microscopy For scanning electron microscope (SEM), the samples were first washed with phosphate buffer solution; then, fixed with 2.5% glutaraldehyde at 4°C overnight, washed with phosphate buffer solution twice; and then fixed in 1% osmium tetroxide solution for 1 hr, and washed twice. The fixed cells were dehydrated by sequential immersion in increasing concentrations of alcohol (30%, 50%, 70%, 80%, 90%, 95% and 100%, V/V), washed twice for every concentration. Lastly, the dehydrated cells were immersed in acetonitrile (50%, 70%, 80%, 90%, 95%, 100%, V/V), 15 min for every concentration, vacuumed, and coated with gold. The observation was conducted using a SEM (5900LV, Hitachi, Japan). For transmission electron microscope (TEM), the anammox bacteria were washed with phosphate buffer solution; then fixed with 2.5% glutaraldehyde at 4°C overnight, washed, and aggregated by agarose pre-embedment. Then, the samples were fixed in 1% osmium tetroxide solution for 1 hr, then dehydrated by sequential immersion in increasing concentrations of acetone (50%, 70%, 80%, 90%, 95% and 100%, V/V), 15 min for every concentration and twice in anhydrous acetone to remove the last traces of water. Finally, the samples were embedded with epoxy resin, sliced into thin sections with a microtome, and examined under a TEM (1200EX, Hitachi, Japan). 1.5.3 DNA extraction, retrieval of 16S rRNA gene sequences and phylogenetic analysis The sludge sample was suspended in 10 mL of DNA extraction buffer (100 mmol/L Tris/HCl, pH 8.0; 100 mmol/L sodium EDTA, pH 8.0; 100 mmol/L sodium phosphate, pH 8.0; 1.5 mol/L NaCl, 1% CTAB) (Schmid et al., 2003). Total genomic DNA was extracted as described previously (Juretschko et al., 1998). The PCR amplification of 16S rRNA genes of members of the Planctomycetales sp. was performed with two groups of primers, the forward primer Pla46F (5 -GACTTGCATGCCTAATCC-3 ) was the same in each group, and the universal reverse primers were 630R (5 -CAKAAAGGAGGTGATCC-3 )

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(Juretschko et al., 1998; Schmid et al., 2003) and 1390R (5 -GACGGGCGGTGTGTACAA-3 ) respectively. PCR was performed with a Gradient cycler (TaKaRa TP600, TaKaRa, Japan). PCR components include DNA 2 μL (about 10 ng), 5× Prime STAR buffer (Mg2+ plus) 10 μL, Pla46F (20 pmol/μL) 0.5 μL, 630R (20 pmol/μL) 0.5 μL, dNTP mixture (2.5 mmol/L respectively) 4 μL, Prime STAR HS DNA polymerase (2.5 U/μL) 0.5 μL, dH2 O 32.5 μL, total 50 μL. PCR operating conditions were described as follows: initial denaturation (98°C, 10 sec), primer annealing (55°C, 15 sec), extending (72°C, 2 min), 35 cycles. The PCR products were connected to pMD19-T Simple Vector with a TaKaRa DNA Ligation kit, then they were converted into Escherichia coli JM109, spread on the plate, and placed overnight at 37°C. Ten positive clones were isolated, and sequencing was done using a Terminator Cycle Sequencing v2.0 kit. The reaction mixtures were analyzed with a Genetic Analyzer (ABI 310, Applied Biosystems, USA).

2 Results and discussion 2.1 Nitrogen removal The performance of the anammox hybrid reactor was monitored for 362 days, shown in Figs. 2 and 3. The influent concentrations of ammonium and nitrite were both elevated from 50 to 270 mg/L. On day 60, the simultaneous removal of NH4 + -N and NO2 − -N was clearly observed, indicating occurrence of the anammox reaction. The nitrate production could serve as an indicator for anammox (Strous et al., 1998), which rose from 0.41 to 73.9 mg/L in the effluent. The HRT was shortened from 12 to 6.1 hr. The nitrogen removal efficiency went up first and then maintained at a level above 80%, and the maximum total NRR of 1.85 kg N/(m3 ·day) was obtained when the NLR was 2.09 kg N/(m3 ·day) on day 362 (HRT, 6.1 hr) (Fig. 3). During day 315–362, outlets I and II were analyzed (above and below the non-woven carrier), including nitrogen concentrations and SS. The ammonium and nitrite Influent NH4+-N Effluent NH4+-N Influent NO2--N

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rescein isothiocyanate (FITC)-labled Amx820 probe (5 AAAACCCCTCTACTTAGTGCCC-3 ) (Schmid et al., 2000) and 4 ,6-diamidino-2-phenylindole (DAPI), and they were purchased from TaKaRa Biotechnology Co., Ltd. (Japan). The sludge samples were fixed overnight in 4% paraformaldehyde phosphate-buffered saline at 4°C. Fixed cells (about 5 μL) were spotted on gelatin-coated slides and allowed to dry in a sterile room temperature space. Hybridization was performed at 46°C for 2 hr. After hybridization, unbound oligonucleotides were removed by rinsing with a washing buffer containing the same components as the hybridization buffer except for the probe. For detection of all bacteria, samples were additionally stained with DAPI. The slides were examined with an Olympus Inverted Microscope (Olympus ZX71, Japan), and digital images of the slides were captured with a digital camera (Nikon D7000, Nikon Corporation, Japan). Image Pro-Plus software was utilized to analyze FISH images.

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concentrations of outlet I were less than outlet II, but the nitrate concentration was higher (Fig. 4). The average ammonium removal efficiencies of outlets I and II with the influent concentration of 270 mg/L were 89.1% and 84.0%, respectively, and the average nitrite removal efficiencies of outlets I and II with the influent concentration of 270 mg/L were 96.0% and 90.6%, respectively. The reaction ratio of the removed nitrite and ammonium through the non-woven carrier was 1.06, close to the report of 1.32 (Strous et al., 1998); the reaction ratio indicated that there were some anammox cells attached to the non-woven carrier. The average total NRR of outlets I and II was 1.61 and 1.49 kg N/(m3 ·day), respectively, and the total NRR of outlet I was 8.1% more than II. The average effluent SS concentrations of outlets I and II were 0.0009 and 0.0035 g/L, respectively (Fig. 5). This showed that the non-woven carrier could catch the anammox cells effectively, which increased the biomass and improved the NRR in the reactor. 2.2 Characteristics of anammox granules The seed granules were gray and black in color, with an average diameter of 0.032 mm (0.02–2.00 mm). During the sludge hydrolysis phase, the granules changed to black with a smelly odor. Then, some sludge near the influent pipe turned light brown during the subsequent phase. After

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the anaerobic ammonium oxidation occurred and became the dominant reaction, the color of the granules gradually changed to red. On day 362, the whole granular sludge had a red color, and the average diameter of sludge granules had increased to 1.510 from 0.032 mm (on day 1). The anammox granules obtained in this study indicated that the sedimentation rate was about 67.8 m/hr, SVI30 of 21.92 mL/g MLVSS at the end of the experiment. The specific anammox activity was up to 0.84 g N/(g MLVSS·day), which was 20% more than that reported in a previous paper (Tang et al., 2011). 2.3 SEM observation The SEM photos of the granular sludge (Fig. 6a) in the hybrid reactor showed a high degree of compactness, each micro-element was tightly integrated with other parts and there was little interspace among them. The micro units inside could interlock with each other, which was in favor of the granular sludge joining tightly and existing stably. Based on observation of the granular anammox sludge, it was concluded that the structure possessed high compactness. SEM photos of the granular sludge (Fig. 6b) showed the sphericity of the cells clearly, and the cellular size was quite uniform, about 0.8–1.2 μm. A few stayed separate, while most tended to grow in clusters. Furthermore, some bacillus were observed, and crowded around anammox cells. 2.4 TEM observation Anammox bacteria have a unique ultrastructure, with an intracytoplasmic membrane that surrounds an organellelike intracytoplasmic compartment, the anammoxosome, which is also the locus of anammox catabolism (Van Niftrik et al., 2004). To confirm the presence of anammoxosomes in the cells, the biomass was inspected with TEM. Thin sections prepared from the reactor running 360 days showed the presence of cells consistent with the anammox structural plan as previously described (Van Niftrik et al., 2008). Some distinct morphotypes of cells with such a cell plan were detected, and they grouped in a distinct microcolony arrangement, closely packed (Fig. 7A) or solitary (Fig.

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Fig. 6 SEM photos of biomass in the hybrid reactor after cultivating 362 days. (a) SEM photos of the granular sludge, 100×; (b) SEM photos of the granular sludge, 10000×.

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Fig. 7 Thin section showing the morphotypes of the groups of cells and one solitary cell. All cells are divided into three separate compartments by individual membranes: the paryphoplasm (p), riboplasm (r), and anammoxosome compartments (a). Besides these compartments, the anammoxosome membrane (am), intracellular iron storage particles (Fe) and glycogen (g) are also observed. (A): a microcolonial group of closely packed cells; (B), (C), (D): the whole anammox structure. Bar = 0.2 μm.

7B, C). Closely fixed cells exhibited only some aspects of the anammox plan, and they included the characteristic and diagnostic anammoxosome compartment, but did not display the classical anammox cell plan including the tubules, nucleoid apposed to anammoxosome membrane, and riboplasm with ribosome-like particles separated from the paryphoplasm at the cell rim by an intracytoplasmic membrane (Fig. 7A). According to the shapes of anammoxosome membranes,

the volumes of cells, and the percentages of anammoxosome compartment compared to the whole cell, three different forms of anammox cells were observed. Some cells had a curved anammoxosome membrane, some cells had an oval anammoxosome membrane (Fig. 7B, C), and some cells had a partly curved, partly oval anammoxosome membrane (Fig. 7C). From the volume of cells and the percentages of anammoxosome compartment compared to the whole cell, cells were round or oval, the cellular diameter

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Fig. 8 FISH analysis of sludge samples from the hybrid reactor on day 15 (a, b), and day 250 (c, d). Green color (a, c) indicates the anammox bacteria hybridized with the FITC-labled Amx820 probe, and blue color indicates (b, d) whole bacteria hybridized with DAPI Fluorescent dye.

was 0.6–1.0 μm, and the percentages of anammoxosome compartment were 51%–85% of the whole cell volume (Fig. 7B, C and D).

was consistent with the past reports on the discovery of anammox bacteria in nature ecosystems or wastewater treatment systems (Kuypers et al., 2003; Dalsgaard et al., 2005). On day 250, the situation in the reactor was quite different. As Fig. 8c describes, anammox bacteria became the dominant population, and Image Pro-Plus software determined the percentage of anammox bacteria to be 51% of total bacteria.

2.5 FISH analysis Sludge samples on day 15 and day 250 were analyzed by the FISH technique. Microbial groups, including anammox bacteria and all bacteria, were investigated in the study. The Amx820 probe was used to target anammox bacteria, and DAPI was used to target all bacteria. The anammox culture was green, confirming the presence of anammox bacteria species as well as the quantity in the reactor. On day 15, there were only a few bacteria hybridized with Amx820, indicating that the portion of anammox bacteria in the reactor (Fig. 8a) was very small; Image Pro-Plus software determined that the percentage of anammox bacteria was 0.75% of total bacteria. Since anammox bacteria grew slowly with a doubling time of about 11 days, a few anammox bacteria from the inoculation of conventional activated sludge was detected at the early stage, which

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2.6 16S rRNA clone library Since a clear assignment of these planctomycetes to anammox organisms was not possible by FISH with existing probes, we applied a Planctomycetales specific 16S rRNA approach to clarify bacterial species. DNA was extracted and 16S rRNA gene sequences of planctomycetes were amplified by PCR with the primers Pla46F and two kinds of reverse primers and cloned. Ten clones of the resulting clone library were randomly sequenced with the reverse primer 630R and almost full length 16S rRNA sequences (average length 700 bases Candidatus Brocadia fulgida (DQ459989) 99 Candidatus Scalindua brodae (AY254883) Candidatus Brocadia sinica (AB565477) 99

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Candidatus Jettenia asiatica (DQ301513) Candidatus Anammoxoglobus propionicus (DQ317601) 100 Candidatus Scalindua brodae (AY254883)

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Candidatus Scalindua sorokinii (AY257181) Candidatus Scalindua wagneri (EF684910) 74

Candidatus Scalindua wagneri (AY254882) 100 Pseudomonas stutzeri (FJ527558) EN 13 Pseudomonas sp. (FJ938156) Thauera aminoaromatica (FJ609701)

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Fig. 9 Phylogenetic tree reflecting the 16S rRNA gene relationships. Showing 10 randomly sequenced clones (EN1, EN2, EN3, EN4, EN5, EN6, EN8, EN9, EN12, EN13) with reverse primer 630R, Candidatus Scalindua brodae, Candidatus Scalindua wagneri, Candidatus Scalindua sorokinii, Candidatus Kuenenia stuttgartiensis, Candidatus Brocadia anammoxidans, other Planctomycetales, and other related organisms. Phylogenetic analyses were performed with p-distance, neighbor joining and 1000 bootstrap replications for bacteria as well as Planctomycetales. For further information about the sequences, please refer to NCBI.

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98 M0607.613-1_5-PLA46F M0607.615-1_7-PLA46F Candidatus Brocadia anammoxidans (AF375994)

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Candidatus Brocadia sinica (GQ175285) M0607.617-1_9-PLA46F Candidatus Brocadia fulgida (DQ459989)

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Candidatus Scalindua brodae (AY257181) 98 Candidatus Scalindua sorokinii (AY257181) Isosphaera pallida (X64372)

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Fig. 10 Phylogenetic tree reflecting the 16S rRNA gene relationships. Showing M0607 X-Pla46F (10 randomly sequenced clones with reverse primer 1390R) and other related organisms. Phylogenetic analyses were performed with neighbor joining and 1000 bootstrap replications for bacteria as well as Plantomycetales.

without primer) were obtained. Subsequent phylogenetic analysis (Fig. 9) showed that four sequences branched extremely close to the known anammox genera and formed a sequence cluster around the anammox species Candidatus Kuenenia sp., and had 100% sequence similarity to the clone (accession No. AF375995). One clone was close to Thauera sp., and had 100% sequence similarity to Thauera aminoaromatica (accession No. FJ609701). One clone formed a sequence cluster around the species Pseudomonas sp., and had 100% sequence similarity to the clone Pseudomonas stutzeri (accession No. FJ527558). Thauera sp. belongs to the family of Rhodocyclaceae, the class of beta-proteobacteria, short rod, which is consistent with the SEM observation (Fig. 6b). It is known to reduce nitrate by denitrification (Philipp et al., 2000). Heterotrophic bacteria are able to oxidize a range of other organic compounds (Hubert et al., 2007). Pseudomonas stutzeri is a denitrifier commonly isolated from both soil (Gamble and Voordouw, 1977) and marine environments (Ward and Cockcroft, 1993), belonging to the class of Gamma-Proteobacteria, which can degrade carbon tetrachloride to carbon dioxide and other inert compounds. It also can degrade phenanthrene and other toxic organic chemicals under aerobic conditions. Thus, the presence of the bacteria, such as Thauera aminoaromatica and Pseudomonas stutzeri, played a positive role in the system; Pseudomonas stutzeri reduced nitrate by denitrification, and improved NRR. In addition, they can consume any oxygen leaking into the reactor, so that the reactor can still maintain anaerobic or anoxic conditions, which is favorable for anammox bacteria (Dapena-Mora et al., 2004). Ten clones of the resulting clone library were randomly sequenced with the reverse primer 1390R. Subsequent phylogenetic analysis showed that the sequences formed two new branches (Fig. 10). The 16S rRNA gene se-

quence similarity of one branch to the closest relative (Candidatus Brocadia anamoxidans) was 52% and the accession number of the 16S rRNA gene is AF375994. The other branch had 89% sequence similarity with the known anammox species Candidatus Brocadia fulgida (accession No. DQ459989). This indicated that the two organisms represented by 16S rRNA gene sequences indeed constituted a new species in the anammox line of descent of the Planctomycetes.

3 Conclusions The anammox process was successfully started up from conventional activated sludge using a hybrid bioreactor within 2 months. Finally the average removal efficiencies of ammonia and nitrite were both over 80%, and the maximum total NRR of 1.85 kg N/(m3 ·day) was obtained on day 362. SEM images clearly showed a high degree of compactness and cell sphericity, and the cell size was quite uniform. TEM photos showed that cells were round or oval, the cellular diameter was 0.6–1.0 μm, and the percentages of anammoxosome compartment were 51%–85% of the whole cell volume. FISH analysis indicated that anammox bacteria became the dominant population in the community (accounting for more than 51% of total bacteria on day 250). Seven planctomycete 16S rRNA gene sequences were present in the 16S rRNA gene clone library generated from the biomass and affiliated to Candidatus Kuenenia stuttgartiensis and a new anammox species. In addition, the role of the non-woven carrier was investigated. The average effluent SS concentrations above and below the non-woven carrier were 0.0009 and 0.0035 g/L, respectively. The non-woven carrier could catch the anammox cells effectively, which stopped biomass loss and improved the NRR in the reactor.

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Journal of Environmental Sciences 2012, 24(6) 1083–1090 / Xiumei Duan et al.

Acknowledgments This work was supported by the Fundamental Research Funds for the Central Universities (No. DUT09RC(3)304), the Key Laboratory of Industrial Ecology and Environmental Engineering, China Ministry of Education (No. KLIEEE-09-09), and the National Natural Science Foundation of China (No. 51008045).

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