J. Microbiol. Biotechnol. (2014), 24(7), 879–887 http://dx.doi.org/10.4014/jmb.1401.01016 Research Article Review jmb

Successful Enrichment of Rarely Found Candidatus Anammoxoglobus propionicus from Leachate Sludge Shu-Chuan Hsu, Yen-Chun Lai, Ping-Heng Hsieh, Pun-Jen Cheng, Suen–Shin Wong, and Chun-Hsiung Hung*

Department of Environmental Engineering, National Chung Hsing University, Taichung 402, Taiwan

Received: January 9, 2014 Revised: March 4, 2014 that mediate the anaerobic oxidation of () have been detected in Accepted: April 3, 2014 natural , as well as various wastewater treatment systems. In this study, sludge from a particular landfill leachate anaerobic treatment system was selected as the incubation seed for anammox microorganism enrichment owing to its possible anammox activity.

First published online Transmission electron microscopy observation, denaturing gradient April 7, 2014 analysis, and cloning/sequencing techniques were applied to identify the diversity of

*Corresponding author anammox microorganisms throughout the incubation. During the early stage of operation, the Phone: +886-4-22840441; diversity of anammox microorganisms was similar to the original complex microbes in the Fax: +886-4-22862589; seed sludge. However, as incubation time increased, the anammox microorganism diversity E-mail: [email protected] within the system that was originally dominated by Candidatus (Ca.) Brocadia sp. was replaced by Ca. Anammoxoglobus propionicus. The domination of Ca. Anammoxoglobus propionicus produced a stable removal of ammonia (70 mg-N/l) and (90 mg-N/l), and the total removal efficiency was maintained at nearly 95%. The fluorescence in situ hybridization results showed that Ca. Anammoxoglobus propionicus was successfully enriched from 1.8 ± 0.6% initially to 65 ± 5% after 481 days of operation. Therefore, the present results demonstrated the feasibility of enriching Ca. Anammoxoglobus propionicus from leachate sludge, even though the original cell count was extremely low. Application of this seldom pISSN 1017-7825, eISSN 1738-8872 found anammox organism could offer an alternative to current ammonia-nitrogen treatment.

Copyright© 2014 by The Korean Society for Microbiology Keywords: Anammox, Ca. Anammoxoglobus propionicus, PCR-DGGE, primer, TEM and

Introduction Ca. Jettenia asiatica, Ca. Kuenenia stuttgartiensis, Ca. , Ca. Scalindua sorokinii, and Ca. Scalindua brodae [2, Anaerobic ammonium oxidation (anammox) was recently 11, 20]. Anammox bacteria are characterized by their developed as a biological wastewater treatment technology extremely slow growth rate; the doubling time of anammox and has also become a potential solution for the removal bacteria ranges from 5 to 30 days [17, 25, 28]. Recent studies of nitrogenous contaminants from wastewater [8, 15]. have also revealed that some of these microorganisms can Microorganisms that catalyze this reaction have been use organic acids as electron donor and undergo anammox identified as anammox bacteria, which are chemolithoautotrophic [6, 11, 26]. microorganisms that conserve energy by oxidizing ammonium Anammox bacteria can be found in many ecosystems, with nitrite as an electron acceptor under anaerobic including agricultural soils, contaminated porous aquifers, conditions. Anammox bacteria compose a distinct, deep freshwater and marine sediments, hot springs, lakes, branching phylogenetic group in the order Planctomycetales; lakeshores, marshes, oxygen minimum zones, and wastewater five genera of anammox bacteria have already been described treatment plants [7, 9, 13, 14, 18, 23]. Yet, different and provisionally named Candidatus (Ca.) Anammoxoglobus anammox species are rarely found in the same , propionicus, Ca. Brocadia anammoxidans, Ca. Brocadia fulgisa, and there are large phylogenetic distances between different

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species as members of Planctomycetales [11]. For example, the anammox bacteria. Sludge collected from a local landfill + Ca. Brocadia anammoxidans and Ca. Kuenenia stuttgartiensis leachate anaerobic treatment system (treating 500 mg-N/l NH4 –N) were found to coexist in a leachate-treating rotating disk was used as the seeding. This landfill is used for the disposal of contactor, and approximately 15% of the nitrite utilized domestic wastes, yet unusually high concentrations of nitrogen during autotrophic growth was converted to [5]. Xiao gas have been measured in its anaerobic tank off-gas, indicating potential anammox activity. For each batch, the reactor was filled et al. [34] also confirmed that Ca. Kuenenia stuttgartiensis with 900 ml of a mineral medium consisting of (NH ) SO (0.778 g), was an anammox microorganism thriving in a landfill 4 2 4 NaNO (0.739 g), KHCO (1.25 g), NaH PO (0.06 g), MgSO ·7H O leachate treated in a sequencing batch reactor. 2 3 2 4 4 2 (0.2 g), CaCl2·2H2O (0.3 g), FeSO4 (0.00625 g), EDTA (0.00625 g), Yapsakli et al. [35] subsequently established that Ca. HCl (1 M, 1 ml), and 1 ml of a trace element solution II [28] per Kuenenia stuttgartiensis is the major nitrogen converter in liter of demineralized water. Once the gas production for each leachate treatment plants. Daverey et al. [4] also demonstrated batch operation stopped completely, a fresh medium was added the simultaneous occurrence of partial , anaerobic to replace the used medium without wasting any biomass. ammonium oxidation, and in a landfill Overall, each batch lasted for an average of 20 days. The leachate treated under a single partially aerated bioreactor, headspace was backfilled at 25 ml/min with 95% Ar-5% CO2 for and indicated that Ca. Kuenenia stuttgartiensis was one of 10 min to maintain anoxic conditions. The pressure of the the dominant species in the reactor. headspace was maintained slightly higher than atmospheric In our previous study, we monitored the composition of pressure. The reactor was then sealed with silicone stoppers. The pH was set at 7 to 8 and then measured and adjusted with biogas from a leachate treatment anaerobic tank located in hydrochloric acid. The temperature was not controlled and set at central Taiwan and found high concentrations of N2 in its + room temperature (27°C to 30°C). The concentrations of NH4 –N, off-gas aside from . This rare phenomenon − − NO2 –N, and NO3 –N were measured using colorimetric methods prompted our interest to examine the possible existence of according to standard procedures [3]. anammox bacteria in that particular system. Initial results confirmed the existence of anammox microorganisms, but Fluorescence In Situ Hybridization (FISH) with low cell counts (data not shown). Accordingly, in the For the FISH analysis, this study used a Cy3-labeled oligonucleotide present study, a laboratory-scale batch reactor designed to probe targeting Ca. Anammoxoglobus propionicus to investigate the operate in an autotrophic anammox mode was operated to anammox bacteria. The hybridizations with fluorescent probes enrich the anammox microorganisms using this leachate were performed as described by Zilles et al. [36]. The total cell sludge as the initial seed. The anammox bacteria in this counts were determined by DAPI staining, plus a probe S-*-Apr- enrichment were characterized, and their physiological 0820-a-A-21 targeting Ca. Anammoxoglobus propionicus and 40% formamide concentration were used for the hybridization experiment characteristics compared with those of previously reported [11]. A total of ten FISH images were taken of the same hybridization anammox bacteria. A regular water quality analysis, sample to determine the average ratio of anammox bacteria to denaturing gradient gel electrophoresis (DGGE), cloning, total bacteria. and transmission electron microscopy (TEM) were also applied to explore the microbial community composition. DNA Extraction, Polymerase Chain Reaction (PCR), and DGGE The total DNA was extracted from each enrichment culture Materials and Methods sample (approximately 1 ml) using an UltraClean Soil DNA Isolation Kit (MOBIO, Solana Beach, USA), following the manufacturer’s Enrichment and Cultivation of Anammox Bacteria instructions. The 16S rRNA gene fragments from the extracted A laboratory-scale batch-mode reactor (blood vase, 900 ml total DNA were then amplified with Taq DNA polymerase culture volume, 100 ml headspace) was used to enrich and culture (GoTAq Green Master Mix; Promega, Madison, USA) using the

Table 1. Summary of 16S rRNA gene-based PCR primers used to detect anammox bacteria in this study. Primer Specificity group Sequence (5’-3’) Target sitea References PLA46FPlanctomycetales GGATTAGGCATGCAAGTC 46-63 [16] Amx368R All anammox bacteria CCTTTCGGGCATTGCGAA 368-385 [22] Amx368FAll anammox bacteria TTCGCAATGCCCGAAAGG 368-385 [22] Amx820R Kuenenia stuttgartiensis/ Brocadia anammoxidans, AAAACCCCTCTACTTAGTGCCC 799-820 [20] Brocadia fulgida a16S rRNA position according to Escherichia coli numbering.

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bacteria and all anammox bacteria specific primer set Pla46F- Amx368R [16, 22], as shown in Table 1. The PCR products were electrophoresed on a 1.5% (w/v) agarose gel. The DGGE was conducted at 60°C in 1× TAE buffer at 80 V for 12 h on a Dcode system (Bio-Rad Laboratories) with a 6% polyacrylamide gel and 20% to 80% denaturant gradient. The gels were stained for 10 min with ethidium bromide and visualized with UV radiation. After visualization under UV transillumination, specific gel bands were excised using a sterilized scalpel. Upon confirmation of the excisions as single bands via a secondary DGGE run, the bands were re- amplified and then sequenced.

Cloning and Phylogenetic Analysis The amplified PCR products were cloned using a TA Cloning Kit (Yeastern Biotech, Taiwan), following the manufacturer’s instructions. Selected PCR products were then further distinguished using DGGE analysis under previously described conditions to obtain the operational taxonomic units (OTUs). All the PCR products with different compositions were subjected to further sequencing (Mission Biotech, Taiwan). The isolated anammox bacterial cultures were identified by comparing their 16S rRNA gene sequences with those in available databases using the BLAST software on the NCBI website [1]. Phylogenetic trees were constructed based on the neighbor-joining method using a Fig. 1. Variation of nitrogen compounds in the batch reactor. molecular evolutionary genetics analysis package (MEGA ver. 4). Each set of points indicates initial and final concentrations for each The robustness of the phylogeny was tested using a bootstrap batch test, ammonium (initial ( ■ ) and final ( □ )), nitrite (initial ( ▲ ) analysis with 1,000 iterations. The isolated and clone sequences and final (△)), and nitrate (initial (◆) and final (◇)). used in this study can be retrieved from the GenBank database under accession number KC862502. During the start-up period (1 to 60 days), the nitrogen removal Transmission Electron Microscopy (TEM) rates fluctuated between 11% and 30%. The removal rates The samples from the reactor were fixed in a 2% glutaraldehyde of ammonia and nitrite reached nearly 100% after 2 months solution in a 0.1% phosphate buffer (pH 6.8 to 7.4) for 2 to 4 h at of operation. An increase in the removal rates of ammonia room temperature. Next, they were fixed with 1% osmium acid and nitrite was accompanied with a significant amount of for 2 to 4 h after cleaning with a phosphate buffer solution (0.1%, pH 6.8 to 7.4). The samples were then dehydrated through a nitrogen gas production. The gas production increased graded series of 50%, 70%, 85%, 95%, and 100% ethanol. Thereafter, from 70 to 100 ml and then to 200 ml per batch the samples were treated with pure ethanol for 30 min, and then (approximately 20 days for one batch). The highest gas treated with a mixture of a coating agent and ethanol [1:1 (v/v)] yield was 300 ml for one batch. Approximately 8 ± 1 to overnight at 4°C. Afterward, the samples were infiltrated using a 13 ± 2 mg-N/l of nitrate was accumulated from day 88 to pure coating agent and left overnight at 70°C. Ultrathin sections of day 286. Thereafter, the system became more stable and the 70 to 90 nm in size were obtained using a Reichert microtome. removal rates of ammonia and nitrite were maintained at These sections were stained with a lead citrate solution and uranyl approximately 100%. The concentration of nitrate during acetate in a 50% saturated ethanol solution for 15 min. Finally, the the final period ranged from 12 ± 9 to 27 ± 9 mg-N/l. The samples were observed under a transmission electron microscope ratios of the average ammonium consumption to nitrite (JEOL JEM-1400, Japan). conversion to nitrate production ranged from 1:1.3:0.3 and 1:1.1:0.5 between day 88 to day 286 and day 286 to day 504, Results and Discussion respectively. The overall stoichiometric nitrate production, nitrite utilization, and ammonium removal ratio also Reactor Operation and Nitrogen Removal During Cultivation agreed with the predicted ratio, 1:1.32:0.26, from a previous The concentration profiles of ammonium, nitrite, and study [25]. As shown in Fig. 1, the concentration of nitrate nitrate from the batch reactor operation are shown in Fig. 1. during the final period suddenly increased to 98 mg-N/l

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on day 312. This result can be attributed to cracking of the were the only two reactions occurring in the system, 108 ml reactor wall, which allowed oxygen to enter the reactor. of nitrogen gas would have been produced, and a final This disturbance was avoided after the reactor was fixed. nitrate concentration of 32 mg-N/l would have been Thus, overall, anammox activity was successfully established obtained. Therefore, the difference between the final during the 500 days of operation when using leachate measured nitrate value of 21 mg-N/l and the calculated sludge as the seed. nitrate value of 32 mg-N/l suggests the existence of a third In biological nitrogen removal, nitrogen compounds can reaction in addition to anammox and denitrification. In be involved in five biological processes; namely, ammonium previous research, it was found that anammox microorganisms oxidation, nitrite oxidation, denitrification, dissimilatory could reduce nitrate to ammonium [10, 29]. Hence, the − nitrate reduction to ammonium (DNRA), and anammox. In 11 mg-N/l gap between the final measured NO3 -N the batch reactor operated in this study, the enrichment concentration (21 mg-N/l) and the modeling results was maintained under anoxic conditions. Thus, nitrification (32 mg-N/l) could be attributed to the consumption of − (including ammonium oxidation and nitrite oxidation) was NO3 -N through dissimilatory nitrite reduction by anammox. − − not involved. Therefore, when estimating the nitrogen If DNRA utilized 1 mol of NO3 -N per mole of NO2 -N or + balance in the reactor, it was assumed that anammox, NH4 -N [10], then an extra 10 ml of N2 gas should have denitrification, and DNRA were all involved in the been obtained through anammox. In this study, approximately conversion of nitrogen. The quantity of nitrogen consumed 120 ml of gas was actually produced, which agreed with by anammox and denitrification was then modeled based the calculated value of 118 ml [105 ml (anammox) + 3 ml on the corresponding stoichiometric equations. The initial (denitrification) + 10 ml (DNRA)]. These stoichiometric + − − feeding concentrations of ammonia, nitrite, and nitrate calculations of NH4 -N, NO2 -N, and the NO3 -N concentrations were 59±1, 78±2, and 20±1 mg-N/l, respectively. The matched well with the corresponding final measured batch initial feeding of chemical oxygen demand (COD) was 28 ± concentrations. Therefore, the results confirmed that the

2 mg/l as O2. After the operation, the final concentrations main nitrogen removal mechanism in the reactor was of ammonia, nitrite, and nitrate were 0.1, 0.1, and 21 ± anammox. 2 mg-N/l, respectively. The TN removal rate was maintained + − − + at 86%, and approximately 120 ml of nitrogen gas was NH4 + 1.32 NO2 + 0.066 HCO3 + 0.13 H → 1.02 N2 + − produced. The final COD value was measured to be 0.26 NO3 + 0.066 CH2O0.5N0.15 + 2.03 H2O(1)

20 ± 2 mg/l as O2. − Thus, the following stoichiometric relationships were C1.6H3.3O1.1N0.02 + 1.6 NO3 → 0.8 N2 + 1.6 CO2 + 1.1 H2O + − assumed for the various biological activities occurring in 0.02 NH3 + 1.6 OH (2) + − the reactor: (i) the molar ratio of NH4 -N : NO2 -N consumed in anammox is 1:1.32, which produces 0.26 mol DGGE Analysis of Enrichment Microbial Community − of NO3 -N, as shown in Eq. (1) [12, 27, 30]; (ii) theoretically, 16S rRNA-based PCR–DGGE technology was also used − denitrification can utilize 1.6 mol of NO3 -N per mole of to investigate the microbial succession of the bacterial COD consumption, as shown in Eq. (2) [32]. The average community during the long-term experimental period values from two batch tests were used for the following using leachate sludge from a local landfill as the seed. The + − modeling. The final concentration of NH4 -N and NO2 -N existence of an anammox bacterial community was verified − was 0.1 mg-N/l after anammox, and 15 mg-N/l of NO3 -N from the PCR-DGGE profile using the primer set Pla46F/ was produced. Therefore, TN changed from 157 mg-N/l to Amx368R targeting members of Planctomycetales [16, 21]. 35.2 mg-N/l (initial nitrate 20 mg-N/L plus 15 mg-N/l was In the seeding sludge, seven to eight PCR-amplified DNA produced); that is, anammox removed approximately 77% bands were detected (Fig. 2). This anammox bacterial TN in the reactor and should produce 105 ml of nitrogen community in the original leachate sludge comprised Ca. gas. COD is used by heterotrophic bacteria as a carbon and Brocadia sp. 40, Ca. Kuenenia sp., and some unidentified energy source during denitrification. Thus, since the anammox microorganisms. After 46 days of operation, most concentration of final COD was 20 mg/l as O2, it was of the Planctomycetales microorganisms were eliminated assumed that 8 mg/l as O2 of the COD was utilized by the from the reactor during the incubation. As shown in Fig. 2, system for denitrification. Consequently, the process only only one clear PCR-DGGE band existed after the first produced 3 ml of nitrogen gas when consuming 3 mg-N/l month of operation. The PCR product of this particular of nitrate. In summary, if anammox and denitrification band was carefully removed from the gel and further

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Fig. 2. DGGE profiles of PCR-amplified DNA fragment (~320 bp) using primers Pla46F/Amx368R.

Fig. 3. DGGE profiles of PCR-amplified DNA fragment (~450 bp) purified. The purified PCR product was sequenced and using primers Amx368F/Amx820R. identified to have 98% similarity to Ca. Brocadia sp. 40 (AM285341.1). This significant change in the Planctomycetales diversity may have been due to the selection effect of the slowly became one of the dominant anammox microorganisms artificial medium or that the initial cell count of Ca. Brocadia throughout the 500-day operation. Ca. Brocadia sp. 40 and sp. was the highest in the seeding sludge. The incubation Ca. Anammoxoglobus propionicus coexisted from day 74 to was continued without changing any operational factor; as day 312. After this period, the only anammox microorganism a result, another clear PCR-DGGE band appeared after 2 existing in the reactor was Ca. Anammoxoglobus propionicus. months of operation and gradually became the only These results agreed with the results from the previous Planctomycetales member throughout the operation (more PCR-DGGE experiments using a more general Planctomycetales than 500 days). The PCR product sequence of this primer set. particular DGGE band was identified to have 100% The anammox microorganisms in the reactor were also similarity to Ca. Anammoxoglobus propionicus (DQ317601.1). explored using different molecular methods. The nitrogen As a further investigation of the anammox microorganism removal rate significantly increased when the bacterial diversity shift, another PCR primer set, Amx368F/Amx820R community diversity shifted from the initial frequently [20, 21], was used to target the 16S rRNA fragment of Ca. found anammox bacteria, Ca. Brocadia sp. 40, Ca. Kuenenia Brocadia sp. and Ca. Kuenenia sp. that are often found in sp., and some unidentified anammox microorganisms, to a leachate sludge samples. Although this primer set was seldom identified Ca. Anammoxoglobus propionicus. Thus, designed for detecting Ca. Brocadia sp. and Ca. Kuenenia sp., after Ca. Anammoxoglobus propionicus became the dominant the results from this study showed that it could also anammox microorganism, stable nitrogen removal was amplify some seldom found anammox microorganisms, achieved, and the total nitrogen (TN) removal efficiency such as Ca. Anammoxoglobus propionicus in this case. Fig. 3 was maintained at 95%. Moreover, the removal rates of shows the PCR-DGGE profile using the second primer set ammonia and nitrite increased from 16% to 92% and from of Amx368F/Amx820R with the same sludge samples 22% to 99.9%, respectively. The reason for this anammox collected during the 500-day operation. A similar diversity community shift is unclear. Ca. Anammoxoglobus propionicus shift was also found when using the second primer set. possibly existed in the original leachate sludge, yet its During the startup stage, Ca. Brocadia sp. 40 was the major viable cell number was too low to be detected. Once the anammox microorganism in the system, with a slight artificial medium was added and the reactor was operated signal of Ca. Brocadia caroliniensis. Ca. Anammoxoglobus in a well-controlled environment, Ca. Anammoxoglobus propionicus appeared in the system on day 74 and then propionicus prevailed over other frequently found anammox

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microorganisms and became the dominant one. The rarely probe selection was based on a previous study by Kartal found anammox microorganism Ca. Anammoxoglobus et al. [11], which suggested that Ca. Anammoxoglobus propionicus is among the five representative anammox propionicus could be hybridized using the probe S-*-Apr- species that have been reported. This bacterium was 0820-a-A-21. The anammox enrichment culture was examined originally found in a laboratory-scale bioreactor in the using FISH after 16 months. The results showed that 65% of presence of ammonium and propionate [11]. Interestingly, the bacterial population was hybridized with the Ca. since Ca. Anammoxoglobus propionicus can reduce nitrate Anammoxoglobus propionicus Cy3-labeled probe S-*-Apr- and/or nitrite to ammonium, this trait could provide Ca. 0820-a-A-21. Ca. Anammoxoglobus propionicus was enriched Anammoxoglobus propionicus a competitive advantage in from 1.8 ± 0.6% (day 46) to 15 ± 3% (day 88) and then to ammonium-limited natural ecosystems [11]. Kartal et al. 65 ± 5% (day 481) (Fig. 4). The definite presence and likely [10] also reported that Ca. Anammoxoglobus propionicus can dominance of Ca. Anammoxoglobus propionicus were identified dominate over other anammox bacteria and heterotrophic in this study. denitrifiers, acquiring most propionate as a supplementary electron donor in the presence of ammonium. To date, only Phylogenetic Diversity of Anammox Microorganisms Using a few studies have reported on Ca. Anammoxoglobus Cloning propionicus being enriched from sludge from wastewater Thirty PCR product clones obtained by applying the treatment plants [11, 33]. Therefore, this study is the first to primer set Amx368F/1492R were randomly selected to report the successful enrichment of Ca. Anammoxoglobus construct a 16S rDNA clone library of the anammox propionicus from leachate with no addition of propionate. bacterial community using sludge collected on day 481. The obtained sequences could be grouped into three OTUs Quantification of Ca. Anammoxoglobus propionicus Using based on more than 99% sequence similarity. The nearly FISH complete 16S rDNA sequence of a representative clone During the incubation, Ca. Anammoxoglobus propionicus from each OTU was analyzed and utilized for the slowly became the predominant anammox microorganism. phylogenetic analysis. The three OTUs were found to be Until now, this microorganism has never been detected affiliated with one phylum. The sequences of AR01 (28 out from leachate sludge. To understand its contribution to of 30 clones), AR02 (1 out of 30 clones), and AR03 (1 out of nitrogen removal, Ca. Anammoxoglobus propionicus was 30 clones) demonstrated 99% similarity to Ca. Anammoxoglobus quantified using hybridization targeting 16S rRNA. The propionicus (Fig. 5). The sequence similarity among the 28

Fig. 4. FISH analysis of anammox bacteria. All bacteria were monitored using DAPI (A, C, and E); Ca. Anammoxoglobus propionicus was hybridized with Cy3-labeled probe Apr820 (B, D, and F). Samples A and B were cultured for 46 days, samples C and D for 88 days, and samples E and F for 481 days. Bar, 20 µm.

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Fig. 5. Phylogenetic tree representing the affiliation of 16S rRNA clone sequences of anammox bacteria with sludge in the reactor. The tree was generated with approximately 1,000 bp of the 16S rRNA gene through the neighbor-joining method. Scale bars = 5% sequence divergence. The values at the nodes are bootstrap values (1,000 times resampling analysis). The NCBI accession numbers are also indicated. clones in OTU AR01 exceeded 99%, indicating that the only membrane-bound intracytoplasmic compartment, known anammox bacterial type present in the reactor was likely to as an anammoxosome [24]. TEM was performed on thin be a strain of Ca. Anammoxoglobus propionicus. The 16S sections prepared from the biomass (collected from the rDNA sequences of AR01 were compared with those of Ca. batch reactor on day 481) containing the anammox organisms. Anammoxoglobus propionicus (NCBI Accession No. DQ317601.1) The anammox species in the leachate seeding system using MEGA4 software. AR01 exhibited an approximately displayed the typical features of anammox bacteria. The 0.78% mismatch with Ca. Anammoxoglobus propionicus in species had a single membrane-bound anammoxosome the database. The AR01 sequences used in this study can be and an anammoxosome membrane-attached nucleoid and retrieved from the GenBank database under Accession No. riboplasm with ribosome-like particles separated from the KC862502. paryphoplasm at the cell rim by an intracytoplasmic membrane (Fig. 6). Previous results also indicated that the TEM Observation of Anammox Bacteria only anammox bacterium in this system is Ca. Anammoxoglobus All previously reported anammox organisms have a propionicus (481 days). The TEM images could be good

Fig. 6. Transmission electron micrograph of thin-sectioned C. Anammoxoglobus propionicus organism. The cells contained the conventional anammox cell components: anammoxosome (A), riboplasm (R) containing a nucleoid (N) opposed to an anammoxosome membrane (M), paryphoplasm (P) separated from the riboplasm by an intracytoplasmic membrane (ICM), and cytoplasmic membrane (CM).

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representatives of the Ca. Anammoxoglobus propionicus DC, USA. found in the leachate. In this study, the average diameter of 4. Daverey A, Su S-H, Huang Y-T, Lin J-G. 2012. Nitrogen Ca. Anammoxoglobus propionicus ranged from 1 to 2µm, removal from opto-electronic wastewater using the simultaneous which agrees with a previous study where the anammox partial nitrification, anaerobic ammonium oxidation and bacterium with the largest diameter (1.1 µm) was Ca. denitrification (SNAD) process in sequencing batch reactor. Bioresour. Technol. 113: 225-231. Anammoxoglobus propionicus [19, 31]. The cells of Ca. 5. Egli K, Fanger U, Alvarez PJJ, Siegrist H, van der Meer JR, Kuenenia stuttgartiensis and Ca. Brocadia fulgida have an Zehnder AJB. 2001. Enrichment and characterization of an average diameter of 800 nm; the cells of Ca. Scalindua are anammox bacterium from a rotating biological contactor slightly larger, with an average cell diameter of 950 nm treating ammonium-rich leachate. Arch. Microbiol. 175: 198- [31]. Thus, since the TEM results showed the features of Ca. 207. Anammoxoglobus propionicus, the anammox bacterium in the 6. Güven D, Dapena A, Kartal B, Schmid MC, Maas B, van de present study was likely a strain of Ca. Anammoxoglobus Pas-Schoonen K, et al. 2005. Propionate oxidation by and propionicus. methanol inhibition of anaerobic ammonium-oxidizing bacteria. In conclusion, based on an anammox community study Appl. Environ. Microbiol. 71: 1066-1071. and TEM observation, a seldom found anammox microorganism 7. Humbert S, Tarnawski S, Fromin N, Mallet M-P, Aragno M, phylogenetically related to Ca. Anammoxoglobus propionicus Zopfi J. 2010. Molecular detection of anammox bacteria in was enriched using leachate sludge as the seeding. Once terrestrial ecosystems: distribution and diversity. Int. Soc. Microb. Ecol. J. 4: 450-454. this Ca. Anammoxoglobus propionicus became the dominant 8. Innerebner G, Insam H, Franke-Whittle IH, Wett B. 2007. anammox microorganism in the reactor, the overall Identification of anammox bacteria in a full-scale nitrogen removal rate increased and remained stable for deammonification plant making use of anaerobic ammonia nearly 500 days. A persistent and stable anammox process oxidation. Syst. Appl. Microbiol. 30: 408-412. was achieved in the system when maintaining the TN feed 9. Jaeschke A, Op den Camp HJM, Harhangi H, Klimiuk A, concentration at 80 to 150 mg-N/l. The average stoichiometric Hopmans EC, Jetten MSM, et al. 2009. 16S rRNA gene and ratio of ammonium consumption to nitrite conversion to biomarker evidence for anaerobic ammonium-oxidizing nitrate production was 1:1.3:0.3. This study proved that the bacteria (anammox) in California and Nevada hot springs. seldom found anammox microorganism Ca. Anammoxoglobus FEMS Microbiol. Ecol. 67: 343-350. propionicus could be enriched from the environment 10. Kartal B, Kuypers MMM, Lavik G, Schalk J, Op den Camp without adding propionate. The potential application of HJM, Jetten MSM, Strous M. 2007. Anammox bacteria this particular anammox microorganism in nitrogen disguised as denitrifiers: nitrate reduction to dinitrogen gas via nitrite and ammonium. Environ. Microbiol. 9: 635-642. removal is worthy of further study. 11. Kartal B, Rattray J, van Niftrik L, van de Vossenberg J, Schmid MC, Webb RI, et al. 2007. Candidatus “Anammoxoglobus Acknowledgments propionicus” a new propionate oxidizing species of anaerobic ammonium oxidizing bacteria. Syst. Appl. Microbiol. 30: 39- The authors would like to thank the National Science 49. Council of the Republic of China, Taiwan, for financially 12. Kuai L, Verstraete W. 1998. Ammonium removal by the supporting this research under Contract Nos. NSC 99-2622- oxygen-limited autotrophic nitrification-denitrification system. E-005-012-CC3 and 100-2622-E-005-004-CC3. Appl. Environ. 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