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Successful Enrichment of Rarely Found Candidatus Anammoxoglobus propionicus from Leachate Sludge
Successful Enrichment of Rarely Found Candidatus Anammoxoglobus propionicus from Leachate Sludge
Journal of Microbiology and Biotechnology. 2014. Jul, 24(7): 879-887
Copyright © 2014, The Korean Society For Microbiology And Biotechnology
  • Received : January 09, 2014
  • Accepted : April 03, 2014
  • Published : July 28, 2014
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About the Authors
Shu-Chuan Hsu
Yen-Chun Lai
Ping-Heng Hsieh
Pun-Jen Cheng
Suen–Shin Wong
Chun-Hsiung Hung
badger@nchu.edu.tw

Abstract
Bacteria that mediate the anaerobic oxidation of ammonium (anammox) have been detected in natural ecosystems, 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. transmission electron microscopy observation, denaturing gradient gel electrophoresis analysis, and cloning/sequencing techniques were applied to identify the diversity of anammox microorganisms throughout the incubation. During the early stage of operation, the diversity of anammox microorganisms was similar to the original complex microbes in the seed sludge. However, as incubation time increased, the anammox microorganism diversity 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 nitrite (90 mg-N/l), and the total nitrogen 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 found anammox organism could offer an alternative to current ammonia-nitrogen treatment.
Keywords
Introduction
Anaerobic ammonium oxidation (anammox) was recently developed as a biological wastewater treatment technology and has also become a potential solution for the removal of nitrogenous contaminants from wastewater [8 , 15] . Microorganisms that catalyze this reaction have been identified as anammox bacteria, which are chemolithoautotrophic microorganisms that conserve energy by oxidizing ammonium with nitrite as an electron acceptor under anaerobic conditions. Anammox bacteria compose a distinct, deep branching phylogenetic group in the order Planctomycetales; five genera of anammox bacteria have already been described and provisionally named Candidatus (Ca.) Anammoxoglobus propionicus, Ca. Brocadia anammoxidans, Ca. Brocadia fulgisa, Ca. Jettenia asiatica, Ca. Kuenenia stuttgartiensis, Ca. Scalindua wagneri, Ca. Scalindua sorokinii, and Ca. Scalindua brodae [2 , 11 , 20] . Anammox bacteria are characterized by their extremely slow growth rate; the doubling time of anammox bacteria ranges from 5 to 30 days [17 , 25 , 28] . Recent studies have also revealed that some of these microorganisms can use organic acids as electron donor and undergo anammox [6 , 11 , 26] .
Anammox bacteria can be found in many ecosystems, including agricultural soils, contaminated porous aquifers, freshwater and marine sediments, hot springs, lakes, lakeshores, marshes, oxygen minimum zones, and wastewater treatment plants [7 , 9 , 13 , 14 , 18 , 23] . Yet, different anammox species are rarely found in the same ecosystem, and there are large phylogenetic distances between different species as members of Planctomycetales [11] . For example, Ca. Brocadia anammoxidans and Ca. Kuenenia stuttgartiensis were found to coexist in a leachate-treating rotating disk contactor, and approximately 15% of the nitrite utilized during autotrophic growth was converted to nitrate [5] . Xiao et al . [34] also confirmed that Ca. Kuenenia stuttgartiensis was an anammox microorganism thriving in a landfill leachate treated in a sequencing batch biofilm reactor. Yapsakli et al . [35] subsequently established that Ca. Kuenenia stuttgartiensis is the major nitrogen converter in leachate treatment plants. Daverey et al . [4] also demonstrated the simultaneous occurrence of partial nitrification, anaerobic ammonium oxidation, and denitrification in a landfill leachate treated under a single partially aerated bioreactor, and indicated that Ca. Kuenenia stuttgartiensis was one of the dominant species in the reactor.
In our previous study, we monitored the composition of biogas from a leachate treatment anaerobic tank located in central Taiwan and found high concentrations of N 2 in its off-gas aside from methane. This rare phenomenon prompted our interest to examine the possible existence of anammox bacteria in that particular system. Initial results confirmed the existence of anammox microorganisms, but with low cell counts (data not shown). Accordingly, in the present study, a laboratory-scale batch reactor designed to operate in an autotrophic anammox mode was operated to enrich the anammox microorganisms using this leachate sludge as the initial seed. The anammox bacteria in this enrichment were characterized, and their physiological characteristics compared with those of previously reported anammox bacteria. A regular water quality analysis, denaturing gradient gel electrophoresis (DGGE), cloning, and transmission electron microscopy (TEM) were also applied to explore the microbial community composition.
Materials and Methods
- Enrichment and Cultivation of Anammox Bacteria
A laboratory-scale batch-mode reactor (blood vase, 900 ml culture volume, 100 ml headspace) was used to enrich and culture the anammox bacteria. Sludge collected from a local landfill leachate anaerobic treatment system (treating 500 mg-N/l NH 4 + –N) was used as the seeding. This landfill is used for the disposal of domestic wastes, yet unusually high concentrations of nitrogen gas have been measured in its anaerobic tank off-gas, indicating potential anammox activity. For each batch, the reactor was filled with 900 ml of a mineral medium consisting of (NH 4 ) 2 SO 4 (0.778 g), NaNO 2 (0.739 g), KHCO 3 (1.25 g), NaH 2 PO 4 (0.06 g), MgSO 4 ·7H 2 O (0.2 g), CaCl 2 ·2H 2 O (0.3 g), FeSO 4 (0.00625 g), EDTA (0.00625 g), HCl (1 M, 1 ml), and 1 ml of a trace element solution II [28] per liter of demineralized water. Once the gas production for each batch operation stopped completely, a fresh medium was added to replace the used medium without wasting any biomass. Overall, each batch lasted for an average of 20 days. The headspace was backfilled at 25 ml/min with 95% Ar-5% CO 2 for 10 min to maintain anoxic conditions. The pressure of the headspace was maintained slightly higher than atmospheric pressure. The reactor was then sealed with silicone stoppers. The pH was set at 7 to 8 and then measured and adjusted with hydrochloric acid. The temperature was not controlled and set at room temperature (27℃ to 30℃). The concentrations of NH 4 + –N, NO 2 –N, and NO 3 –N were measured using colorimetric methods according to standard procedures [3] .
- Fluorescence In Situ Hybridization (FISH)
For the FISH analysis, this study used a Cy3-labeled oligonucleotide probe targeting Ca. Anammoxoglobus propionicus to investigate the anammox bacteria. The hybridizations with fluorescent probes were performed as described by Zilles et al . [36] . The total cell counts were determined by DAPI staining, plus a probe S-*-Apr-0820-a-A-21 targeting Ca. Anammoxoglobus propionicus and 40% formamide concentration were used for the hybridization experiment [11] . A total of ten FISH images were taken of the same hybridization sample to determine the average ratio of anammox bacteria to total bacteria.
- DNA Extraction, Polymerase Chain Reaction (PCR), and DGGE
The total DNA was extracted from each enrichment culture sample (approximately 1 ml) using an UltraClean Soil DNA Isolation Kit (MOBIO, Solana Beach, USA), following the manufacturer’s instructions. The 16S rRNA gene fragments from the extracted total DNA were then amplified with Taq DNA polymerase (GoTAq Green Master Mix; Promega, Madison, USA) using the bacteria and all anammox bacteria specific primer set Pla46FAmx368R [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℃ 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 s ing le bands via a secondary DGGE run, the bands were reamplified and then sequenced.
Summary of 16S rRNA gene-based PCR primers used to detect anammox bacteria in this study.
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a16S rRNA position according to Escherichia coli numbering.
- Cloning and Phylogenetic Analysis
The amplified PCR products were c loned 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 molecular evolutionary genetics analysis package (MEGA ver. 4). The robustness of the phylogeny was tested using a bootstrap analysis with 1,000 iterations. The isolated and clone sequences used in this study can be retrieved from the GenBank database under accession number KC862502.
- Transmission Electron Microscopy (TEM)
The samples from the reactor were fixed in a 2% glutaraldehyde solution in a 0.1% phosphate buffer (pH 6.8 to 7.4) for 2 to 4 h at room temperature. Next, they were fixed with 1% osmium acid 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 graded series of 50%, 70%, 85%, 95%, and 100% ethanol. Thereafter, the samples were treated with pure ethanol for 30 min, and then treated with a mixture of a coating agent and ethanol [1:1 (v/v)] overnight at 4℃. Afterward, the samples were infiltrated using a pure coating agent and left overnight at 70℃. Ultrathin sections of 70 to 90 nm in size were obtained using a Reichert microtome. These sections were stained with a lead citrate solution and uranyl acetate in a 50% saturated ethanol solution for 15 min. Finally, the samples were observed under a transmission electron microscope (JEOL JEM-1400, Japan).
Results and Discussion
- Reactor Operation and Nitrogen Removal During Cultivation
The concentration profiles of ammonium, nitrite, and nitrate from the batch reactor operation are shown in Fig. 1 . During the start-up period (1 to 60 days), the nitrogen removal rates fluctuated between 11% and 30%. The removal rates of ammonia and nitrite reached nearly 100% after 2 months of operation. An increase in the removal rates of ammonia and nitrite was accompanied with a significant amount of nitrogen gas production. The gas production increased from 70 to 100 ml and then to 200 ml per batch (approximately 20 days for one batch). The highest gas yield was 300 ml for one batch. Approximately 8 ± 1 to 13 ± 2 mg-N/l of nitrate was accumulated from day 88 to day 286. Thereafter, the system became more stable and the removal rates of ammonia and nitrite were maintained at approximately 100%. The concentration of nitrate during the final period ranged from 12 ± 9 to 27 ± 9 mg-N/l. The ratios of the average ammonium consumption to nitrite 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, respectively. The overall stoichiometric nitrate production, nitrite utilization, and ammonium removal ratio also agreed with the predicted ratio, 1:1.32:0.26, from a previous study [25] . As shown in Fig. 1 , the concentration of nitrate during the final period suddenly increased to 98 mg-N/l on day 312. This result can be attributed to cracking of the reactor wall, which allowed oxygen to enter the reactor. This disturbance was avoided after the reactor was fixed. Thus, overall, anammox activity was successfully established during the 500 days of operation when using leachate sludge as the seed.
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ation of nitrogen compounds in the batch reactor. Each set of points indicates initial and final concentrations for each batch test, ammonium (initial (■) and final (□)), nitrite (initial (▲) and final (△)), and nitrate (initial (◆) and final (◇)).
In biological nitrogen removal, nitrogen compounds can be involved in five biological processes; namely, ammonium oxidation, nitrite oxidation, denitrification, dissimilatory nitrate reduction to ammonium (DNRA), and anammox. In the batch reactor operated in this study, the enrichment was maintained under anoxic conditions. Thus, nitrification (including ammonium oxidation and nitrite oxidation) was not involved. Therefore, when estimating the nitrogen balance in the reactor, it was assumed that anammox, denitrification, and DNRA were all involved in the conversion of nitrogen. The quantity of nitrogen consumed by anammox and denitrification was then modeled based on the corresponding stoichiometric equations. The initial feeding concentrations of ammonia, nitrite, and nitrate were 59 ± 1, 78 ± 2, and 20 ± 1 mg-N/l, respectively. The initial feeding of chemical oxygen demand (COD) was 28 ± 2 mg/l as O 2 . After the operation, the final concentrations of ammonia, nitrite, and nitrate were 0.1, 0.1, and 21 ± 2 mg-N/l, respectively. The TN removal rate was maintained at 86%, and approximately 120 ml of nitrogen gas was produced. The final COD value was measured to be 20 ± 2 mg/l as O 2 .
Thus, the following stoichiometric relationships were assumed for the various biological activities occurring in the reactor: (i) the molar ratio of NH 4 + -N : NO 2 - -N consumed in anammox is 1:1.32, which produces 0.26 mol of NO 3 - -N, as shown in Eq. (1) [12 , 27 , 30] ; (ii) theoretically, denitrification can utilize 1.6 mol of NO 3 - -N per mole of COD consumption, as shown in Eq. (2) [32] . The average values from two batch tests were used for the following modeling. The final concentration of NH 4 + -N and NO 2 - -N was 0.1 mg-N/l after anammox, and 15 mg-N/l of NO 3 - -N was produced. Therefore, TN changed from 157 mg-N/l to 35.2 mg-N/l (initial nitrate 20 mg-N/L plus 15 mg-N/l was produced); that is, anammox removed approximately 77% TN in the reactor and should produce 105 ml of nitrogen gas. COD is used by heterotrophic bacteria as a carbon and energy source during denitrification. Thus, since the concentration of final COD was 20 mg/l as O 2 , it was assumed that 8 mg/l as O 2 of the COD was utilized by the system for denitrification. Consequently, the process only produced 3 ml of nitrogen gas when consuming 3 mg-N/l of nitrate. In summary, if anammox and denitrification were the only two reactions occurring in the system, 108 ml of nitrogen gas would have been produced, and a final nitrate concentration of 32 mg-N/l would have been obtained. Therefore, the difference between the final measured nitrate value of 21 mg-N/l and the calculated nitrate value of 32 mg-N/l suggests the existence of a third reaction in addition to anammox and denitrification. In previous research, it was found that anammox microorganisms could reduce nitrate to ammonium [10 , 29] . Hence, the 11 mg-N/l gap between the final measured NO 3 - -N concentration (21 mg-N/l) and the modeling results (32 mg-N/l) could be attributed to the consumption of NO 3 - -N through dissimilatory nitrite reduction by anammox. If DNRA utilized 1 mol of NO 3 - -N per mole of NO 2 - -N or NH 4 + -N [10] , then an extra 10 ml of N 2 gas should have been obtained through anammox. In this study, approximately 120 ml of gas was actually produced, which agreed with the calculated value of 118 ml [105 ml (anammox) + 3 ml (denitrification) + 10 ml (DNRA)]. These stoichiometric calculations of NH 4 + -N, NO 2 - -N, and the NO 3 - -N concentrations matched well with the corresponding final measured batch concentrations. Therefore, the results confirmed that the main nitrogen removal mechanism in the reactor was anammox.
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- DGGE Analysis of Enrichment Microbial Community
16S rRNA-based PCR-DGGE technology was also used to investigate the microbial succession of the bacterial community during the long-term experimental period using leachate sludge from a local landfill as the seed. The existence of an anammox bacterial community was verified from the PCR-DGGE profile using the primer set Pla46F/ Amx368R targeting members of Planctomycetales [16 , 21] . In the seeding sludge, seven to eight PCR-amplified DNA bands were detected ( Fig. 2 ). This anammox bacterial community in the original leachate sludge comprised Ca. Brocadia sp. 40, Ca. Kuenenia sp., and some unidentified anammox microorganisms. After 46 days of operation, most of the Planctomycetales microorganisms were eliminated from the reactor during the incubation. As shown in Fig. 2 , only one clear PCR-DGGE band existed after the first month of operation. The PCR product of this particular band was carefully removed from the gel and further purified. The purified PCR product was sequenced and 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 artificial medium or that the initial cell count of Ca. Brocadia sp. was the highest in the seeding sludge. The incubation was continued without changing any operational factor; as a result, another clear PCR-DGGE band appeared after 2 months of operation and gradually became the only Planctomycetales member throughout the operation (more than 500 days). The PCR product sequence of this particular DGGE band was identified to have 100% similarity to Ca. Anammoxoglobus propionicus (DQ317601.1).
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DGGE profiles of PCR-amplified DNA fragment (~320 bp) using primers Pla46F/Amx368R.
As a further investigation of the anammox microorganism diversity shift, another PCR primer set, Amx368F/Amx820R [20 , 21] , was used to target the 16S rRNA fragment of Ca. Brocadia sp. and Ca. Kuenenia sp. that are often found in leachate sludge samples. Although this primer set was designed for detecting Ca. Brocadia sp. and Ca. Kuenenia sp., the results from this study showed that it could also amplify some seldom found anammox microorganisms, such as Ca. Anammoxoglobus propionicus in this case. Fig. 3 shows the PCR-DGGE profile using the second primer set of Amx368F/Amx820R with the same sludge samples collected during the 500-day operation. A similar diversity shift was also found when using the second primer set. During the startup stage, Ca. Brocadia sp. 40 was the major anammox microorganism in the system, with a slight signal of Ca. Brocadia caroliniensis. Ca. Anammoxoglobus propionicus appeared in the system on day 74 and then slowly became one of the dominant anammox microorganisms throughout the 500-day operation. Ca. Brocadia sp. 40 and Ca. Anammoxoglobus propionicus coexisted from day 74 to day 312. After this period, the only anammox microorganism existing in the reactor was Ca. Anammoxoglobus propionicus . These results agreed with the results from the previous PCR-DGGE experiments using a more general Planctomycetales primer set.
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DGGE profiles of PCR-amplified DNA fragment (~450 bp) using primers Amx368F/Amx820R.
The anammox microorganisms in the reactor were also explored using different molecular methods. The nitrogen removal rate significantly increased when the bacterial community diversity shifted from the initial frequently found anammox bacteria, Ca. Brocadia sp. 40, Ca. Kuenenia sp., and some unidentified anammox microorganisms, to a seldom identified Ca. Anammoxoglobus propionicus . Thus, after Ca. Anammoxoglobus propionicus became the dominant anammox microorganism, stable nitrogen removal was achieved, and the total nitrogen (TN) removal efficiency was maintained at 95%. Moreover, the removal rates of ammonia and nitrite increased from 16% to 92% and from 22% to 99.9%, respectively. The reason for this anammox community shift is unclear. Ca. Anammoxoglobus propionicus possibly existed in the original leachate sludge, yet its viable cell number was too low to be detected. Once the artificial medium was added and the reactor was operated in a well-controlled environment, Ca. Anammoxoglobus propionicus prevailed over other frequently found anammox microorganisms and became the dominant one. The rarely found anammox microorganism Ca. Anammoxoglobus propionicus is among the five representative anammox species that have been reported. This bacterium was originally found in a laboratory-scale bioreactor in the presence of ammonium and propionate [11] . Interestingly, since Ca. Anammoxoglobus propionicus can reduce nitrate and/or nitrite to ammonium, this trait could provide Ca. Anammoxoglobus propionicus a competitive advantage in ammonium-limited natural ecosystems [11] . Kartal et al . [10] also reported that Ca. Anammoxoglobus propionicus can dominate over other anammox bacteria and heterotrophic denitrifiers, acquiring most propionate as a supplementary electron donor in the presence of ammonium. To date, only a few studies have reported on Ca. Anammoxoglobus propionicus being enriched from sludge from wastewater treatment plants [11 , 33] . Therefore, this study is the first to report the successful enrichment of Ca. Anammoxoglobus propionicus from leachate with no addition of propionate.
- Quantification of Ca. Anammoxoglobus propionicus Using FISH
During the incubation, Ca. Anammoxoglobus propionicus slowly became the predominant anammox microorganism. Until now, this microorganism has never been detected from leachate sludge. To understand its contribution to nitrogen removal, Ca. Anammoxoglobus propionicus was quantified using hybridization targeting 16S rRNA. The probe selection was based on a previous study by Kartal et al . [11] , which suggested that Ca. Anammoxoglobus propionicus could be hybridized using the probe S-*-Apr-0820-a-A-21. The anammox enrichment culture was examined using FISH after 16 months. The results showed that 65% of the bacterial population was hybridized with the Ca. Anammoxoglobus propionicus Cy3-labeled probe S-*-Apr-0820-a-A-21. Ca. Anammoxoglobus propionicus was enriched from 1.8 ± 0.6% (day 46) to 15 ± 3% (day 88) and then to 65 ± 5% (day 481) ( Fig. 4 ). The definite presence and likely dominance of Ca. Anammoxoglobus propionicus were identified in this study.
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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.
- Phylogenetic Diversity of Anammox Microorganisms Using Cloning
Thirty PCR product clones obtained by applying the primer set Amx368F/1492R were randomly selected to construct a 16S rDNA clone library of the anammox bacterial community using sludge collected on day 481. The obtained sequences could be grouped into three OTUs based on more than 99% sequence similarity. The nearly complete 16S rDNA sequence of a representative clone from each OTU was analyzed and utilized for the phylogenetic analysis. The three OTUs were found to be affiliated with one phylum. The sequences of AR01 (28 out of 30 clones), AR02 (1 out of 30 clones), and AR03 (1 out of 30 clones) demonstrated 99% similarity to Ca. Anammoxoglobus propionicus ( Fig. 5 ). The sequence similarity among the 28 clones in OTU AR01 exceeded 99%, indicating that the only anammox bacterial type present in the reactor was likely to be a strain of Ca. Anammoxoglobus propionicus . The 16S rDNA sequences of AR01 were compared with those of Ca. Anammoxoglobus propionicus (NCBI Accession No. DQ317601.1) using MEGA4 software. AR01 exhibited an approximately 0.78% mismatch with Ca. Anammoxoglobus propionicus in the database. The AR01 sequences used in this study can be retrieved from the GenBank database under Accession No. KC862502.
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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.
- TEM Observation of Anammox Bacteria
All previously reported anammox organisms have a membrane-bound intracytoplasmic compartment, known as an anammoxosome [24] . TEM was performed on thin sections prepared from the biomass (collected from the batch reactor on day 481) containing the anammox organisms. The anammox species in the leachate seeding system displayed the typical features of anammox bacteria. The species had a single membrane-bound anammoxosome and an anammoxosome membrane-attached nucleoid and riboplasm with ribosome-like particles separated from the paryphoplasm at the cell rim by an intracytoplasmic membrane ( Fig. 6 ). Previous results also indicated that the only anammox bacterium in this system is Ca. Anammoxoglobus propionicus (481 days). The TEM images could be good representatives of the Ca. Anammoxoglobus propionicus found in the leachate. In this study, the average diameter of Ca. Anammoxoglobus propionicus ranged from 1 to 2 μm, which agrees with a previous study where the anammox bacterium with the largest diameter (1.1 μm) was Ca. Anammoxoglobus propionicus [19 , 31] . The cells of Ca. Kuenenia stuttgartiensis and Ca. Brocadia fulgida have an average diameter of 800 nm; the cells of Ca. Scalindua are slightly larger, with an average cell diameter of 950 nm [31] . Thus, since the TEM results showed the features of Ca. Anammoxoglobus propionicus , the anammox bacterium in the present study was likely a strain of Ca. Anammoxoglobus propionicus .
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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).
In conclusion, based on an anammox community study and TEM observation, a seldom found anammox microorganism phylogenetically related to Ca. Anammoxoglobus propionicus was enriched using leachate sludge as the seeding. Once this Ca. Anammoxoglobus propionicus became the dominant anammox microorganism in the reactor, the overall nitrogen removal rate increased and remained stable for nearly 500 days. A persistent and stable anammox process was achieved in the system when maintaining the TN feed concentration at 80 to 150 mg-N/l. The average stoichiometric ratio of ammonium consumption to nitrite conversion to nitrate production was 1:1.3:0.3. This study proved that the seldom found anammox microorganism Ca. Anammoxoglobus propionicus could be enriched from the environment without adding propionate. The potential application of this particular anammox microorganism in nitrogen removal is worthy of further study.
Acknowledgements
The authors would like to thank the National Science Council of the Republic of China, Taiwan, for financially supporting this research under Contract Nos. NSC 99-2622-E-005-012-CC3 and 100-2622-E-005-004-CC3.
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