Evaluation of oxygen adaptation and identification of functional bacteria composition for anammox consortium in non-woven biological rotating contactor

Bioresource Technology 99 (2008) 8273–8279

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Evaluation of oxygen adaptation and identification of functional bacteria composition for anammox consortium in non-woven biological rotating contactor Sitong Liu a, Fenglin Yang a,*, Yuan Xue a, Zheng Gong a, Huihui Chen a, Tao Wang a, Zhencheng Su b a Key Laboratory of Industrial Ecology and Environmental Engineering, MOE, School of Environmental and Biological Science and Technology, Dalian University of Technology, Dalian 116024, PR China b Institute of Applied Ecology, Chinese Academy of Sciences, Shenyang 110016, PR China

a r t i c l e

i n f o

Article history: Received 3 February 2008 Received in revised form 4 March 2008 Accepted 4 March 2008 Available online 24 April 2008 Keywords: Anammox Nitrosomonas eutropha Dissolved oxygen Bacteria composition Biological rotating contactor

a b s t r a c t In this study, the anammox consortium was found to adapt to the wastewater containing dissolved oxygen (DO), as the DO was gr\adually increased. Batch tests indicated the maximum aerobic ammonium þ 1 oxidizing activity of the consortium was 1:38 mmolNH4 —Nðg VSSÞ1 day , which played key roles in the oxygen consumption process; the maximum anaerobic ammonium oxidizing activity was slightly þ 1 decreased after long-term oxygen exposure, but only from 21:23 mmolNH4 —Nðg VSSÞ1 day to þ 1 20:23 mmolNH4 —Nðg VSSÞ1 day . Microbiological community analysis identified two strains similar to Nitrosomonas eutropha were responsible for oxygen consumption, which were able to exist in the autotrophic anaerobic condition for long periods and protect anammox bacteria Planctomycetales from the influence of oxygen. Microbiological composition analysis showed Nitrosomonas and Planctomycetales approximately accounted for 10% and 70% of the bacteria, respectively. The possibility of cultivation anammox consortium in presence of DO will lead to substantial savings of energy and resource in the industrial application. Ó 2008 Elsevier Ltd. All rights reserved.

1. Introduction Anammox is a new technology developed recently in which ammonium is converted to N2 with nitrite as electron acceptor under anoxic condition (van de Graaf et al., 1996). Compared with the conventional biological nitrogen removal process, it is a novel, promising, low-cost alternative (Abma and Schultz, 2007; van Loosdrecht and Jetten, 2001; Tsushima et al., 2007) and has many advantages, e.g., no requirement for external carbon sources, low oxygen demand, minimized surplus sludge and reduced CO2 emissions. The investigation of anammox is an important revolution in the nitrogen removal theory and has a huge application foreground in the nitrogen removal technology. The anammox process is a technique which can treat with wastewater contained much ammonium and little organic material, such as sludge digestor effluents. However, the stringent metabolism conditions and extremely slow growth rate of the anammox bacteria have restricted their application to pilot-scale plants (Strous et al., 1998), although some significant works have been successfully done to apply anammox process on actual industrial wastewater (Waki et al., 2007; Dong and Tollner, 2003). The anammox reaction is easily inhibited by oxygen and nitrite. Very low oxygen levels (>0.04 mg L1) inhibit reversibly (Strous et al., * Corresponding author. Tel.: +86 411 8470 6172; fax: +86 411 8470 6171. E-mail address: [email protected] (F. Yang). 0960-8524/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2008.03.006

1998) and high nitrite concentrations (>100 mg L1) inhibit irreversibly (Strous et al., 1999) the anaerobic ammonium oxidizing activity. The influence of oxygen on the anammox process has been investigated in several experiments and the results indicated that only when all the oxygen was removed from the reactor by vigorously flushing with inert gases, the conversion of ammonium and nitrite resumed, thus indicating that the anammox activity in these enrichment cultures is only possible under strict anoxic conditions (Jetten, 2001). In the view of applicability on semi-industrial scale, the anammox consortium must cope with the variable and harsh conditions of wastewater treatment compared with the optimal laboratory condition. Efforts to enlarge the application range of anammox process in industrial utilization have been done over the past years. For example, the combination of anammox and denitrification processes was studied aiming to treat high-ammonium wastewater contained COD (Chamchoi et al., 2007), and then the possibilities of cultivating anammox consortium under low ammonium-fed (Pathak et al., 2007) or low temperature conditions (Dosta et al., 2007) were also investigated. Up to day, one of other problems remained to be solved in practical utilization of anammox process is the adaptation of dissolved oxygen (DO) contained in the influent or reactor, namely the possibility of cultivating anammox consortium under oxygen conditions, which should be evaluated for the industrialization of anammox process in order to save high cost in the maintenance of absolutely anaerobic condition.

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A promising strategy to avoid the DO influence is to enrich some oxygen-consumed bacteria for creating the anammox’s anaerobic niches. However, the cultivation of anammox consortium was often in strictly autotrophic, oxygen-absence conditions. Even though the DO can be supplied in the influent, the concentration is much lower than that in some other processes equipped with oxygen-offered devices. So under this oxygen-lack condition, it is difficult to successfully enrich such oxygen-consumed bacteria and make them take action in the anammox consortium. Actually, there is few kinds of bacteria which can sustain the nutrition lack condition of the anammox consortium for a long time and the diversity of the anammox consortium composition is lower compared with that of some other biological nitrogen removal processes. Whereas, the consistent presence of some oxic nitrifiers in the anammox reactors (Jetten et al., 1999) or anammox biofilms (Schmid et al., 2000) confirms that although nitrifiers may not be enriched under anoxic conditions, they can at least survive. Moreover, it has been known that many of the aerobic ammonium oxidizers, such as some strains belonging to Nitrosomonas, are facultatively anaerobic (Kuenen and Jetten, 2001) and have anaerobic metabolisms. They can use a variety of electron donors (hydrogen, pyruvate, and ammonium) for the reduction of nitrite (Poth and Focht, 1985; Schmidt and Bock, 1998; Bock et al., 1995) under anaerobic condition and produce NO, N2O and N2 (Poth and Focht, 1985; Poth, 1986). In sharp contrast to the anammox, they have more versatile metabolism. The highest anaerobic ammonium oxidizing activity is 25 times lower than þ 1 that of anammox ð55 nmolNH4 —Nðmg proteinÞ1 min Þ (Kuenen and Jetten, 2001), but high enough to survive in prolonged periods of oxygen-absence. For the multiplicate metabolism, the possible long-term existence of some Nitrosomonas strains in anammox consortium has the potential to provide the biotechnological foundation to facilitate the DO adaptation of the anammox consortium. In this work, we have targeted the DO questions for anammox consortium in feasibility studies with wastewater in laboratory scale. The objectives of this paper were to assess the DO adaptation of anammox consortium and the corresponding nitrogen removal performance. DO levels might also have a definite impact on the functional microbial community structure in the reactor, so in this study, members of the functional bacteria were detected and enumerated using the recent developing molecular techniques such as polymerase chain reaction (PCR)-denaturing gradient gel electrophoresis (DGGE) and fluorescence in situ hybridization (FISH). The possibility of DO adaptation in cultivation anammox consortium will lead to substantial savings of energy and resource to the treatment of high-ammonium wastewater.

2. Methods

Fig. 1. Scheme of the NRBC reactor: (1) influent tank; (2) influent pump; (3) horizontal shaft; (4) rotating contactor; (5) rotating device; (6) disc; (7) opening for sample collection; (8) outlet; (9) heater tank; (10) thermometer; (11) cycling pump; (12) aerating hole; (13) thermostatic jacket.

maintain anaerobic condition and covered to protect the bacteria from light and algal growth. The hydraulic retention time (HRT) was fixed at 6 h by a peristaltic pump. 2.2. Synthetic wastewater and inoculum The synthetic wastewater fed to the NRBC reactor in this experiment mainly contained ammonium and nitrite in the form of (NH4)2SO4 and NaNO2, the amounts of which varied depending on the applied load. The composition of the mineral medium was as specially described by van de Graaf et al. (1996). The pH of the synthetic wastewater was adjusted to 8.0 ± 0.1 by 1 M HCl and 1 M Na2CO3 before providing to the reactor. The anammox consortium for inoculation was taken from a laboratory scale anammox up-flow column reactor (Furukawa et al., 2003; Liu et al., 2008). The initial biomass concentration in the reactor was about 0.8 g VSS L1. 2.3. Analytical procedures The concentrations of nitrogen compounds were measured according to standard methods, as set out by the American Public  Health Association (APHA, 1995). NHþ 4 —N and NO2 —N were mea sured colorimetrically, NO3 —N was measured spectrophotometrically. TN was determined by the TOC analyzer equipped with a total nitrogen-measuring unit (TOC-VCPH, Shimadzu). The pH measurement was done using a digital, portable pH meter, DO measurement was done using a digital, portable DO meter (YSI, Model 55, USA). The volatile suspended solids (VSS) were determined to calculate the biomass concentration according to the standard methods (APHA, 1995).

2.1. Non-woven rotating biological contactor reactor 2.4. Operation of the NRBC system The non-woven rotating biological contactor (NRBC) reactor configuration used in the experiment is given in Fig. 1. Non-woven porous polyester coated with a pyridinium-type polymer (Japan Vilene, US patent 5,185,415; 1993) was used as the discs to enhance the attachment performance for its good adsorption characteristics. Ten discs were mounted (10 cm diameter of one disc, 0.5 cm thickness, 1 cm interspace) on a horizontal shaft by fixing with stainless steel. The discs were rotated at 0.5 rpm to mix the substrate with 100% submergence of the disc surface area. The cylindrical reactor equipped with a thermostatic jacket (maintained at 35 °C) made of perspex has an effective volume of 1.7 L. The reactor and feed vessels were all sealed tightly in order to

The experiment was divided into two periods. In period I (day 0–50), the reactor was operated under absolute anaerobic condition by flushing nitrogen gas in the influent. In period II (day 50– 100), the system was turned to oxygen condition, namely the oxygen concentration in the influent was gradually increased by flushing nitrogen gas with gradual decreasing fluxes, until no need of any nitrogen gas. Samples were taken every 2 days and analyzed   for NHþ 4 —N, NO2 —N, NO3 —N and total nitrogen (TN). At the beginning of the experiment and at the end of each the period, the compositions of the biofilm were analyzed with PCR-DGGE and FISH technologies.

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2.5. Batch experiments Batch experiments were performed under the aerobic and anaerobic condition, respectively. They were done by cutting the non-woven carrier in the reactor for activity measurement. Biomass concentration at the beginning of each batch test was around 0.1 g VSS L1. The carrier was washed with distilled water for three times to remove the background media. Concentration  (100 mg L1) of NHþ 4 —N, NO2 —N and other mineral media were diluted to 100 mL with distilled water and added into the 150 mL serum bottles for anaerobic assays. Concentration (100 mg L1) of   NHþ 4 —N or NO2 —N ðNO3 —NÞ and other mineral media were added into the 150 mL Erlenmeyer flasks for aerobic assays. For the anaerobic batch tests, oxygen was removed from the mixed liquor by flushing with nitrogen gas for 10 min and the serum bottles were sealed tightly with rubber caps to avoid any influent of external O2. For the aerobic batch tests, the flasks were shaken at 100 rpm to keep fully aerobic condition for measuring the maximum aerobic activity of the consortium. All the bottles and flasks were incubated at 35 °C. Liquid samples were collected using syringes with needles for monitoring the ammonium and nitrite concentrations over time. All incubations were repeated at three different places in the NFBR system to calculate the average value. 2.6. Polymerase chain reaction amplification and denaturing gradient gel electrophoresis The genomic DNA extraction was performed as previously described by Lakay et al. (2007). The amplification of beta-subdivision of aerobic ammonium oxidizers DNA was performed with the primers 189fAB plus 189fC/654r (Kowalchuk et al., 1998), the amplification of aerobic nitrite oxidizers DNA was performed with the primers PRBA 338f/NIT3r and PRBA338f/Ntspa0685r (Regan et al., 2002), and the amplification of anammox bacteria DNA was performed with the primers 40f/518r (Neef et al., 1998). A 40-nucleotide GC-clamp was added to the forward primer 341f at the 50 -end (50 -CGCC-30 ) to improve the detection of sequence variation in amplified DNA fragments by subsequent DGGE. All the products obtained were subjected to DGGE analysis. DGGE was performed at 60 °C with a D-CODE System Universal Mutation (Bio-Rad Laboratories) according to the manufacture’s instruction. The PCR products were applied on a DGGE gel of 6% polyacrylamide with a linear denaturing gradient ranging from 40% to 60% (100% denaturing gradient contains 7 M urea and 40% formamide). Electrophoresis was run at a constant voltage of 100 V for 17 h in 1  TAE buffer. Subsequently, the gels were stained with SYBR Gold (TaKaRa, China) in 1  TAE buffer for 40 min and gel digital images were obtained using the Gel Doc 2000 System (Bio-Rad Laboratories). 2.7. Sequencing of 16SrRNA gene and phylogenetic analysis Each gel slice that contained an obvious DNA band was excised and placed in a 1.5 ml Eppendorf tube, incubated with TE buffer at

4 °C for 12 h. Then the second PCR was carried out. After amplification, the PCR products were again analyzed by DGGE to confirm their electrophoretic mobility to the fragment from which they were excised. The PCR products using the corresponding 16SrDNA primers with no ‘‘GC” clamp were purified with a PCR purification kit (Dalian, TaKaRa), and used as template DNA in a cycle sequencing reaction with a BigDye Terminator V3.1 in Sangon Company (Shanghai, China). Sequencing of 16SrRNA fragments was carried out with a Sequencing System ABI PRISM 3730 (Applied Biosystems). The obtained sequences were compared with the reference microorganisms available in the GenBank by BLAST search (Altschul et al., 1990). 2.8. Fluorescence in situ hybridization The composition and spatial structure of the microbial community were analyzed by FISH in this study. The probe name, the labeling dye, the target site (16SrDNA positions), the target organism, and the optimal formamide concentration in the hybridization buffers were reported in Table 1. Probes were purchased as fluorophores Cy3, Cy5 and FITC labeled from TaKaRa Company (Dalian, China). Hybridizations were performed on 4% (w/v) paraformaldehyde-fixed biofilm samples. For image acquisitions, an epifluorescence microscope (OlympusBX51, Japan) was used together with the standard software package delivered with the instrument (version 4.0). 3. Results and discussion 3.1. Operation of NRBC Performance of the NRBC system in terms of various nitrogen removal rates is shown in Fig. 3. In period I, both the influent and the reactor were strictly kept anoxic condition, so the nitrogen removal performance under anoxic condition was able to be investigated. There was an exponential increase of TN removal rate, which arrived 2.1 kg N m3 day1 at the end of the steady-operation phase. The temperature, pH and DO concentration in the reactor was kept at 35 °C, 8–8.2, <0.04 mg L1, respectively. The pH value in effluent was significantly higher than that in the influent, namely the consumption of acidity results in pH increase in the anammox process (Hoa et al., 2006). Based on the calculation of total discs surface, the NH4+–N surface removal rate of 26 kg N m2 day1 was obtained. It is much higher than that previously reported (Pynaert et al., 2003), owing to the excellent attachment performance of non-woven carrier. In period II, the possibility of cultivating anammox culture when oxygen existed in the influent was investigated. However, it is a failure to abruptly make the culture adapt the oxygen condition without aerating any nitrogen gas in the influent. The DO concentrations both in the influent and in the reactor were kept at 8 mg L1. Neither the ammonium oxidizing activity nor nitrogen removal rate could be obtained in such system, as present in Fig. 2. As we known, the anammox bacteria have strict metabolism

Table 1 Oligonucleotide probes used for FISH in this study Probe name

Labeling dye

Formamide (%)

Target site (16SrDNA positions)

Target organism

Reference

Eub338 Eub338-II Eub338-III Amx820 Nit3 Nsv443 Neu653

FITC FITC FITC Cy5 Cy3 Cy3 Cy3

0 0 0 40 40 30 40

GCTGCCTCCCGTAGGAGT GCAGCCACCCGTAGG TGT GCT GCC ACC CGT AGG TGT AAA ACC CCT CTA CTT AGT GCCC CCTGTGCTCCATGCTCCG CCG TGA CCG TTT CGT TCCG CCC CTC TGC TGC ACT CTA

Eubacteria Eubacteria Eubacteria Anammox Nitrobacter Nitrosospira Nitrosomonas

Amann et al. (1990) Daims et al. (1999) Daims et al. (1999) Schmid et al. (2000) Wagner et al. (1996) Gieseke et al. (2001) Wagner et al. (1995)

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requirement and the anaerobic ammonium oxidizing activity was so reversibly inhibited by high DO concentration. Moreover, the abundant NO 2 —N in the influent, which could not be in time consumed by the inactive anammox bacteria, would do a great harm to the whole bacteria consortium and affect their biological activities. A step-wise adaptation method was applied in the next experiment. The DO concentration in the influent was step-wise improved, from 1 mg L1 to 5 mg L1, and finally arrived at 8 mg L1—without aerating any nitrogen gas during the experimental period, which was controlled by the decreased flux of nitrogen gas aerated into the influent. In this way, the values of DO concentration in the reactor remained below 0.04 mg L1 most of time, indicating that some kinds of bacteria could consume oxygen and make the reactor anoxic. The production of NO 3 —N was used to express the growth of anammox (van de Graaf et al., 1996), from which we could see that the growth was slow but of no stop (in Fig. 3). This steady nitrogen removal stage with TN removal rate

of 2–2.3 kg N m3 day1 could be kept, suggesting the successful adaptation of DO for anammox consortium. From the Fig. 3, we  could see the ratio of NHþ 4 —N consumption to NO2 —N consumption was slightly increased, compared with that in the absolutely anaerobic condition. It seemed that, some aerobic nitrifiers could  oxidize NHþ 4 —N to NO2 —N and so keep the biofilm anaerobic,  þ while NO2 —N and NH4 —N could be converted by anammox bacteria to N2. However, as a reason of the limited offered oxygen, only dissolved in the influent, the aerobic ammonium oxidizing by nitrifiers was quite limited according to the nitritation parameter. So in this process, there was still some surplus NHþ 4 —N in the effluent, leaving the NO 2 —N as limited substrate. However, this assumption needed to be further validated. This system has been tested and demonstrated stable effluent quality and nitrogen removal rate without the necessary DO control. Then it is can be deduced that it is possible to make anammox consortium to adapt the oxygen condition. In this process, the method of a gradually increase of DO concentration in the influent

    þ Fig. 2. Time courses of NHþ 4 —N, NO2 —N, NO3 —N removal rates and water quality of NH4 —N, NO2 —N, NO3 —N in the influent and effluent in the NRBC reactor before or after DO was abruptly introduced.

    þ Fig. 3. Time courses of NHþ 4 —N, NO2 —N, NO3 —N removal rates and water quality of NH4 —N, NO2 —N, NO3 —N in the influent and effluent in the NRBC reactor before or after DO was gradually increased.

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is a key operational strategy. There would be some coupling reactions in this process: some oxygen-consumed bacteria in the anammox consortium played an important role on protecting anammox bacteria from the effect of oxygen. Simultaneously, the anammox bacteria converted the NO 2 —N, and relieved the inhibition effect of high NO —N concentration to the consortium. How2 ever, it is also strange that the failure of abruptly oxygen introduction. Further study is necessary to investigate this uncommon aerobic and anaerobic reaction and validate the functional bacteria catalyzing this process. 3.2. Aerobic and anoxic experiments with NRBC biomass Sludge samples were collected from NRBC reactor on day 50 (sample A) and day 100 (sample B) to analyze aerobic activity and anaerobic activity by batch tests. The maximum specific anaerobic ammonium oxidizing activity of sample B was a little lower 1 1 ð20:23 mmolNHþ day Þ, compared with that of sam4 —Nðg VSSÞ þ 1 1 ple A ð21:23 mmolNH4 —Nðg VSSÞ day Þ. A possible explanation was that when oxygen existed, the comparatively lower content of the effective functional bacteria capable of anaerobic ammonium oxidizing contributed to the slightly lower anaerobic ammonium oxidizing activity. In the batch-mode aerobic incubation, there is an obvious aerobic ammonium oxidizing activity of sample B and 1 1 the value was 1:38 mmolNHþ day . For sample A, 4 —N ðg VSSÞ the aerobic activity was so low in the first few days that it is difficult to be measured. Afterwards, the activity value was increased 1 1 gradually and arrived at 0:78 mmolNHþ day after 4 —N ðg VSSÞ aerobic cultivation for 5 days. Actually, the microbiological composition was similar between these two samples as suggested by FISH analysis. Then it was confirmed that a period time was in deed required for the aerobic nitrifiers to adapt the new oxygen condition or change the metabolism approach. This experimental result could also explain the necessary step-wise increase of DO for the adaptation of anammox consortium. No nitrite oxidizing activity was identified in this aerobic batch experiment. Another argument was the potential participation of aerobic denitrifiers in the consortium in the oxygen consumption process. In another aerobic batch experiment, the aerobic denitrification activity was tested using the nitrite or nitrate as substrates when added ATU as an inhibiter for nitrifiers. The negative results of the experiments indicated no aerobic denitrifiers in the consortium. From this point, it became sure that the aerobic ammonium oxidizing reaction and anaerobic ammonium oxidizing reaction were the mainly functional reactions for the whole process. In order to obtain the exact biotechnological potential of these reactions, the microbiological community in the NRBC reactor was measured by PCR-DGGE analysis of the DNA directly extracted in the different operational periods in NRBC.

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of one band exhibited 99% similarity with the recently described anaerobic ammonia-oxidizing group KSU-1 (AB057453.1) within the Planctomycetales (Fujii et al., 2002). Another two obtainable major bands had the maximum similarity of 99% and 98% with an unknown uncultured Planctomyces sp. (AY555652.1). Sequences of these two strains independent of all the known anaerobic ammonium oxidizers may represent novel anaerobic ammonium oxidizers groups within the Planctomycetales. Despite the differences in sampling times, samples revealed similar DGGE profiles, suggesting similar community composition of functional Planctomycetes bacteria at these stages of system performance. Specific amplification of 16SrRNA fragment of beta-proteobacterial aerobic ammonium oxidizing bacteria (AOB) and separation of mixed PCR samples by DGGE analysis were combined to investigate the AOB community composition in the consortium. Several bands were detected and the band pattern was not agreed with each other as shown in Fig. 4. The existence of AOB in the consortium thereafter was confirmed. The one obtainable major band of AOB community showed high similarity of 94% to the genus Nitrosomonas eutropha (AJ298739) affiliated with the class proteobacteria, while the other obtainable major band of bacteria community had 99% similarity also to an uncultured N. eutropha (AY123795). Surprisingly, even though oxygen was introduced to the reactor, no change was found, indicating the similar community composition at these stages of system performance. The anammox consortium used in this experiment has been kept under autotrophic anaerobic conditions for many years, from which we

3.3. Denaturing gradient gel electrophoresis and phylogenetic analysis In this study, DNA fragments with the expected size were amplified using primer sets 40f GC/518r, 189fAB plus 189fC GC/ 654r, PRBA 338f GC/NIT3r and PRBA338f GC/Ntspa0685r. DGGE analysis on days 0, 50 and 100 of the operation was applied to evaluate the microbial succession of functional bacteria community. The existence of anammox bacteria appeared evident from the DGGE profile of bacteria DNA amplified by PCR using the primers set 40f GC/518r. The analysis of microbial community by PCRDGGE clarified that Planctomycetes related organisms were highly enriched. In the DGGE fingerprints in Fig. 4, the sample showed three strongly stained bands along with some other minor stained bands. The three dominant DGGE bands were all closely related to a member of Planctomycetales, which was revealed by the phylogenetic analysis based on partial 16SrRNA gene sequences. Sequence

Fig. 4. DGGE analysis of PCR-amplified 16S rRNA gene fragments of biofilm samples in NRBC reactor.

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could conclude that these microorganisms similar to N. eutropha have the ability to sustain the strict condition and coexist with anammox bacteria for a very long period. Because of the failures in PCR amplification using the primers sets PRBA 338f GC/NIT3r or PRBA338f GC/Ntspa0685r, no aerobic nitrite oxidizers were indicated in the consortium. A possible explanation is that neither the Nitrospira nor the Nitrobacter could exist in the biofilm and sustain long-term anaerobic condition. 3.4. Fluorescence in situ hybridization The recent development of molecular biology techniques such as FISH has enabled scientists to overcome the limitation of conventional techniques based on cultivation (Sinha and Annachhatre, 2007). To elucidate the composition and spatial structure of the microbial community in the biofilm, the FISH technique was applied on day 0, day 50 and day 100 of the operation in this study. No indications were found in this study for the existence of Nitrosospira using probe Nsv443 or Nitrobacter using probe Nit3, which correlated nicely with that proposed by the PCR-DGGE analysis. FISH analysis showed that some AOB existed and belonged to Nitrosomonas group, reacting with the Neu653 probe. Moreover, strong hybridization signals with Amx820 probe was found, which was the characteristic of anammox cells. For more detailed localization, sectional imaging of samples using a confocal laser scanning microscope was performed using FITC labeled Eub338 (Eub338, Eub338-II and Eub338-III), Cy3 labeled Neu653 and Cy5 labeled Amx820 as hybridization probes. We performed the FISH experiment in different places of the biofilm. According to the experimental average result, we deduced that Nitrosomonas existed and accounted for about 10% of consortium, and most of the rest (about 70% of the consortium) were anammox bacteria. In the whole operation, nearly no increase proportion of Nitrosomonas was found, even under this oxygen condition. It certainly could be confirmed for the supplied oxygen was so low that it was impossible to be a quick growth level for Nitrosomonas. There was considerable interest in another important observation, which was the formation of special spatial structure of the biomass community after long-term oxygen exposure. We could see that for most of the anammox bacteria congeries, they were enclosed by the Nitrosomonas bacteria. In other words, most of the Nitrosomonas bacteria were distributed in the edge of the anammox congeries. This special spatial structure indeed benefited for this DO adaptation process and the further protection to anammox bacteria from the potential existence of oxygen. In the CANON or SNAP processes, some coupling effects have been built between two bacterial populations: Nitrosomonas-like aerobic ammonium oxidizing bacteria and Planctomycete-like anammox bacteria. The ammonium oxidizers oxidize ammonium to nitrite, consume oxygen and so create anoxic conditions the anammox bacteria need. The produced nitrite is utilized with the remainder of the ammonium by anammox bacteria and converted into nitrogen gas (Nielsen et al., 2005; Lieu et al., 2006). However, there are some differences between the CANON or SNAP processes and this DO adaptation process. In the processes like CANON or SNAP, the influent contains (NH4)2SO4 only. The aerobic nitrifiers account for about 40–50% of the bacteria community, which are enough to carry out the partial nitrification of (NH4)2SO4 to NaNO2 to provide the necessary substrates for anammox bacteria. However, in this study, the influent does not only contain (NH4)2SO4, but also NaNO2. The aerobic nitrifiers are so little (accounts for 10% of the microbiological community) that they cannot provide the necessary NaNO2 for the subsequent anammox reaction. Moreover, there are only few kinds of bacteria belonging to Nitrosomonas can exist in the anammox consortium for long-terms and prepare to carry out the oxygen consumption process.

4. Summary When oxygen contained in the influent, the NHþ 4 —N and NO —N could also be successfully removed by anammox consor2 tium with the steady high nitrogen removal rate of 2– 2.3 kg N m3 day1, while the step-wise increase of DO concentration in the influent was a key operational strategy, for a period of time was required for the functional oxygen-consumed bacteria to convert the metabolism approach. In this nitrogen removal process, aerobic nitrifiers similar to the N. eutropha were the mainly functional oxygen consumption community, and so protected the anammox bacteria belonging to Planctomycetale from the effect of oxygen. Simultaneously, the anammox bacteria converted the  NO 2 —N, and relieved the inhibition effect of high NO2 —N concentration to the consortium. In this way, some coupling reactions were built up, which offered a great biotechnological potential for this anammox process under oxygen condition. Interestingly, the bacteria strains similar to N. eutropha are specialized ammonium oxidizers existing in anammox consortium under strictly autotrophic anaerobic condition for long-terms and so provide the key biotechnological potential to facilitate this DO adaptation. The special spatial structure that N. eutropha enwrapped the Planctomycetale was also beneficial for this oxygen adaptation process. In this way, N. eutropha provided further protection actions for Planctomycetale from the effect of oxygen. From this point, it is verified that the cultivation of the anammox consortium is not restricted to completely anaerobic condition. The N. eutropha were the functional bacteria coexisting with anammox bacteria in anammox consortium. In the further industrial application, some aerobic ammonium oxidizers N. eutropha can be added to the anammox consortium to achieve this oxygen-contained anammox cultivation. The N. eutropha have ability to co-exist with anammox bacteria in the anammox consortium. Moreover, the microbiological composition of this anammox consortium will keep steady during all the operational phase, even under the introduction of oxygen. So the additional N. eutropha will be considered as a simple and convenient approach to validate the oxygen adaptation process for anammox consortium. The possibility of cultivation anammox consortium in presence of DO will offer a great future potential for savings of energy and resource for efficient nitrogen removal from wastewater in the industrial application. Given the low-costs of our system, a full-scale implementation of anammox process is to be expected in the near future and the relevant research about the applicability of anammox process, coping with the variable and harsh conditions of wastewater treatment on semi-industrial scale is underway. Acknowledgements We would like to thank Professor Kenji Furukawa for kindly supplying the anammox bacteria in Faculty of Engineering, Kumamoto University, Kumamoto, Japan.

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