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In situ probing of microbial activity within anammox granular biomass with microelectrodes
Journal of Bioscience and Bioengineering VOL. xx No. xx, 1e7, 2015 www.elsevier.com/locate/jbiosc
In situ probing of microbial activity within anammox granular biomass with microelectrodes Yongtao Lv,1 Kai Ju,1 Lei Wang,1, * Ting Sun,1 Rui Miao,1 Xudong Wang,1 Fan Wei,1 and Siqing Xia2 School of Environmental and Municipal Engineering, Xi’an University of Architecture and Technology, Xi’an 710055, China1 and College of Environmental Science and Engineering, Tongji University, Shanghai 200092, China2 Received 23 April 2015; accepted 29 August 2015 Available online xxx
An anaerobic rotating biological contactor was fed with inorganic synthetic wastewater for anammox. Besides bioL L film, granular biomass with average diameter of approximately 5 mm formed. NHD 4 , NO2 , NO3 and pH microelectrodes were used to probe microbial activity in situ within the granules. At a sufficient substrate concentration, the anammox reaction was observed in the upper layer of granules, and the most active zone was found to be in the surface of 200e400 mm. The in situ anammox activity increased with increasing substrate concentration, and a maximum ammonium consumption rate of 83.3 mmol cmL3 hL1 was obtained at an ammonium concentration of 1000 mmol LL1. Under an ammonium-limited condition, denitrification activity was observed in the inner layer, and the most active zone was limited to 700e1000 mm. This study revealed that denitrification bacteria coexisted with anammox bacteria within inorganic anammox granules. Ó 2015, The Society for Biotechnology, Japan. All rights reserved. [Key words: Anammox; Denitrification; In situ microbial activity; Active zone; Microelectrodes]
Nitrogen removal from wastewater has become an important part of the overall treatment of wastewater because of the significant effect of nitrogen compounds on the groundwater environment (1). However, some wastewater rich in NHþ 4 but poor in biodegradable organic carbon (for example, digested sludge supernatant and landfill leachate) is difficult to treat with the application of a conventional nitrificationedenitrification process. In such cases, anaerobic ammonium oxidation (anammox) has been found to be an alternative process for ammonium removal in the absence of organic matter and oxygen (2). In this process, autotrophic anammox bacteria oxidize ammonium to N2 using nitrite (NO 2 ) as an electron acceptor under strict anoxic conditions. The stoichiometry of this reaction proposed by Strous et al. (3) is shown in Eq. 1. þ NHþ 4 þ 1.32NO2 þ 0.066HCO3 þ 0.13H / 1.02N2 þ 0.26NO3 þ 2.03H2O þ 0.066CH2O0.5N0.15 (1)
To supply nitrite as an electron acceptor and realize an autotrophic nitrogen removal process, anammox is often combined with nitritation. The combined process can reduce operation costs by nearly 40% and is considered an economic and promising way of treating ammonium-rich wastewater (4). Anammox bacteria are mostly anaerobic autotrophs and thus sensitive to concentrations of dissolved oxygen (DO) and organic matter (5,6). However, wastewater is a complex ecosystem;
* Corresponding author. Tel./fax: þ86 29 82202729. E-mail address: [email protected] (L. Wang).
therefore, multiple bacteria usually coexist in an anammox system. Kumar and Lin (4) demonstrated that anammox and denitrification were responsible for the removal of nitrogen from low-C/N wastewater. Kindaichi et al. (6) found that ammonium-oxidizing bacteria (AOB), nitrite-oxidizing bacteria (NOB) and anammox bacteria coexisted in an anammox system (6). The biomass populations and nitrogen removal performance have been well studied by fluorescence in situ hybridization analysis and stoichiometry analysis (5,7e9). However, there is limited knowledge about substrate diffusion and consumption inside bacterial aggregates, which is essential for clarifying the microbial reaction and improving process performance. Microelectrodes, which are needle-shaped devices with sensitive tips, have been demonstrated to be the most suitable tools for the in situ profiling of substrate concentrations within bacterial aggregates (10e12). Studies have investigated the spatial distributions of microbial activity. Gieseke et al. (11) studied substrate transport through biofilm and determined the size of an active zone. Li and Bishop (12) investigated the microprofiles of substrate concentrations within activated sludge flocs and offered an optimization strategy. Satoh et al. (13) and Xiao et al. (14) studied variations in nitrification and denitrification activity within biofilm under different conditions. However, only a few studies have investigated in situ activities of multiple bacteria in anammox aggregates using microelectrodes (6,15,16). A better understanding of the in situ microbial activity will allow further optimization of the reactor. In our previous study (17), an anaerobic rotating biological contactor (AnRBC) was used for anammox and removed more than 90% of total nitrogen (TN) from influent. Besides biofilm attaching to the surface of the discs, we observed the formation
1389-1723/$ e see front matter Ó 2015, The Society for Biotechnology, Japan. All rights reserved. http://dx.doi.org/10.1016/j.jbiosc.2015.08.015
Please cite this article in press as: Lv, Y., et al., In situ probing of microbial activity within anammox granular biomass with microelectrodes, J. Biosci. Bioeng., (2015), http://dx.doi.org/10.1016/j.jbiosc.2015.08.015
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TABLE 1. In situ microbial activity experiments. Concentration (mmol L
No.
1 2 3 4 5
MATERIALS AND METHODS
1
)
NHþ 4
NO 2
NO 3
100 500 1000 100 0
120 600 1200 200 600
0 0 0 0 0
of granules. The objectives of the present study were to identify the spatial distribution of microbial activity within the granular biomass and to investigate the effect of the substrate concentration on microbial activity. To this end, the microbial community present was firstly investigated using polymerase chain reaction (PCR) and denatured gradient gel electrophoresis (DGGE) analysis. And then, pH, NHþ 4 , NO2 and NO3 microelectrodes were used to measure microprofiles under different substrate concentrations. All results obtained from a microscopic view provide valuable information for better understanding the reactor performance.
Experimental set-up Anammox performance was investigated using an AnRBC with a net liquid capacity of 6.2 L that has been operated for more than 7 years (17). The AnRBC has 13 polyvinyl chloride discs, with a total surface area of 0.32 m2 and 87% submersion of the disc surface. The rotation speed of the discs was controlled at 1.3e1.5 r min1. The hydraulic retention time was kept at 1 d. The reactor was maintained at 40 1 C using a water-jacket. Synthetic wastewater The synthetic wastewater used in this study mainly contained NaHCO3, NH4Cl, NaNO2 and trace elements described by van de Graaf et al. (18). The concentrations of NHþ 4 -N and NO2 -N were kept in the range 200e280 mg L1. The pH value of influent was maintained between 7.5 and 8.0. Chemical analysis The concentration of NHþ 4 -N was determined according to standard methods of APHA (19). NO 3 -N and NO2 -N were determined using an ion chromatograph (Metrohm 761, Metrohm, Switzerland). The DO level was measured with a digital portable HI9147 Oxy-check (Hanna, Romania). DNA extraction, PCR-DGGE, cloning, and sequencing The total DNA was extracted using a bacterial genomic mini extraction kit (Sangon, China). To identify the genus of AnAOB, specific primers of AMX818F and AMX1066R were used for the PCR amplification (20). To investigate the community structure, universal primers F357 (with GC clamp) and R518 were used for the PCR amplification (21). For DGGE analysis, the PCR products were purified using a gel midi purification kit (Sangon, China) and
FIG. 1. Time course of the nitrogen removal performance of the AnRBC.
FIG. 2. Morphology (A) and Scanning electron microscopy images (B) of the ANAMMOX granules.
Please cite this article in press as: Lv, Y., et al., In situ probing of microbial activity within anammox granular biomass with microelectrodes, J. Biosci. Bioeng., (2015), http://dx.doi.org/10.1016/j.jbiosc.2015.08.015
VOL. xx, 2015
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separated using DGGE in 8% (wt/vol) polyacrylamide gels containing a linear gradient of 30%e60% denaturant employing a DCode Universal Mutation Detection System (Bio-Rad, USA). Prominent DGGE bands in the gel were excised and dissolved in 100 mL of 1 TE buffer at 4 C overnight. A 1 mL volume of TE solution as a template was reamplified with the primer pairs (without the GC clamp) using the same method as described previously, followed by purification using a purification kit (Sangon, China) and cloning in a pMD19-T plasmid vector system (Takara, Japan). Sequencing was performed using an ABI 3730 DNA sequencer offered by a commercial service (Sangon, China). All the sequences obtained were retrieved from the GenBank database by employing the BLAST alignment tool at the National Center for Biotechnology Information (NCBI). Microsensor measurements Microprofiles of pH and the NHþ 4 , NO2 and NO 3 concentrations within the bacterial aggregates were obtained using micro electrodes. Microelectrodes for measuring pH, NHþ 4 , NO2 and NO3 with tip diameters of approximately 15 mm were manufactured in our laboratory and their performances were confirmed to be stable (22). Before making microprofile measurements, the microelectrodes were prepared, calibrated and operated as described by Satoh et al. (23) and Okabe et al. (24). Although the AnRBC was a biofilm reactor, granular biomass with an average diameter of around 5 mm also formed in our study. Granules were sampled from the inlet of the reactor, and a chamber was used for microsensor measurements (10). After sampling, a single granule was placed gently just above the nylon net in the chamber, and then fixed by two needles. To obtain steady-state profiles, the granule was left for at least 1 h in preincubation before profile measurements were taken. Measurements were made at least three times with each microelectrode. To investigate the in situ activity of anammox bacteria and other bacteria, substrates with different concentrations were used to measure microprofiles (Table 1). Different granules were used at three levels of ammonium concentrations. To remove the batch-to-batch variation, granules with diameter of 5 mm were selected for microprofile measurements. The DO concentration of this medium was kept below 0.05 mg L1 through continuous purging with N2.
Fluxes and rates calculations The following basic assumptions will be made for fluxes and rates calculations: (i) No substrate consumption or production occur in the bulk liquid; (ii) the granules are spherical in shape and uniform in size; (iii) only radial diffusion transport is considered and is described by Fick’s law. Firstly, simulated curves were obtained by fitting the concentration profile using the software Sensor Trace PRO. Secondly, diffusive fluxes are calculated according to Fick’s first law (25): Jn ¼ Ds
dCn C Cn1 ¼ Ds nþ1 drn rnþ1 rn1
(2)
where Jn is the flux at point n (mmol m2 s1), Ds is the effective diffusion coefficient (m2 s1), dCn/drn is the concentration gradient at point n (mmol m3 m1), C is the simulated substrate concentration (mmol m3), and r is the distance from the granule center (m). Values of 1.23 109, 1.1 109 and 1.5 109 m2 s1 were used for diffusion coefficients of nitrite, ammonium and nitrate, respectively (12)). Finally, the net volumetric consumption or production rates (Rn, mmol m3 s1) of NHþ 4 , NO2 and NO3 in each layer n were calculated from the steady-state concentration profiles using Fick’s second law of diffusion (26): Rn ¼
2 2 4prnþ1 Jnþ1 4prn1 Jn1 4 pr 3 3 nþ1
4 pr 3 3 n1
¼ 3
2 2 rnþ1 Jnþ1 rn1 Jn1 3 rnþ1
! (3)
3 rn1
RESULTS AND DISCUSSION Performance of the AnRBC The laboratory-scale AnRBC has been operated in anammox for more than 7 years. During the period of this study, inorganic synthetic wastewater was fed to the reactor. The nitrogen removal performance is shown in Fig. 1.
TABLE 2. The microbial community from the AnRBC. Primers AMX818F/AMX1066R F357/R518
FIG. 3. DGGE analysis of total bacteria in the AnRBC. Detailed information on the bands 1e5 is presented in Table 2.
Closest relative
Identity (%)
Candidatus Kuenenia stuttgartiensis Niabella sp. Sphingobium xenophagum Bellilinea sp. Bellilinea sp. Burkholderiales
100 100 100 96.3 92 100
Band e 1 2 3 4 5
Please cite this article in press as: Lv, Y., et al., In situ probing of microbial activity within anammox granular biomass with microelectrodes, J. Biosci. Bioeng., (2015), http://dx.doi.org/10.1016/j.jbiosc.2015.08.015
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During 100 days of operation, the influent TN concentration was 491.2 23.9 mg L1, the AnRBC removed 91.4% 1.6% of TN, and 0.5 kg m3 d1 of maximum TN removal volume load was obtained. Nitrite and ammonium were removed simultaneously, and the effluent concentrations of nitrite and ammonia were 2.6 2.6 and 2.3 2.8 mg L1, respectively. In contrast, the nitrate concentration increased slightly, from 29.9 5.9 mg L1 in influent to 37.2 6.1 mg L1 in effluent. Nitrate was also detected in influent, it might be mainly caused by the insufficient reagent purity which used in this experiment. At an influent nitrite concentration of 200e280 mg L1, no inhibition of anammox biomass activity was observed in our system, which was in accordance with the result of Cho et al. (27). These results demonstrate that the stratified granules or biofilm biomass tolerated a higher NO 2 concentration than did the homogenized biomass owing to limited diffusion. Because of the complex wastewater quality, multiple bacteria usually coexist in an anammox reactor, e.g., AOB, NOB and denitrifying bacteria. In the present anammox system, the moler ratio of consumed nitrite to ammonium was 1.1 0.1, and the moler ratio of produced nitrate to consumed ammonium was 0.03 0.02. Both values were lower than the ratios in Eq. 1, which demonstrated the presence of another nitrogen removal pathway.
J. BIOSCI. BIOENG., ratio of 1.2:1. Three media having gradient ammonium concentrations of 100, 500 and 1000 mmol L1 were used. The concentration profiles were measured at least three times, and the average profiles are presented in Fig. 4AeC. The simulated curves
Sludge morphology and structure of the microbial community Besides biofilm attaching to the surface of the discs, granular biomass formed in the reactor, especially at the inlet of the reactor. The average diameter of the granules was approximately 5 mm (Fig. 2A), and aggregates of spherical bacteria were observed on the surfaces of the granules with a scanning electron microscope (Fig. 2B). Anammox bacteria are sensitive to environmental conditions (e.g., substrate concentration, DO, pH), and they thus likely grow into stratified granules or biofilm. Anammox granular biomass has been reported in many reactors, such as the upflow anaerobic sludge bed reactor (28,29), upflow granular reactor (27), sequencing batch reactor (8,30,31) and membrane reactor (32). However, granule formation in an AnRBC biofilm reactor has not yet been reported. In our previous study (17), anammox bacteria accounted for approximately 80% of the total biomass, both in granules and biofilms. Therefore, in this study, the AnRBC is a hybrid system in which both attached and suspended sludge coexist. Further studies are needed to clarify the contributions of the two morphological structures of sludge to the nitrogen removal efficiency). Around 7 years and 2 months after the start-up of anammox, the microbial community was investigated using PCR and DGGE analyses. Using the primers AMX818F and AMX1066R, PCR products of 16SrDNA with full length of 241 bp were obtained. Analysis of the DNA sequence revealed that the AnAOB belonged to the genus of Candidatus Kuenenia stuttgartiensis. Using universal primers F357 (with a GC clamp) and R518, five discrete DGGE bands were excised (see Fig. 3), and the DNA was eluted and sequenced. The DNA sequence analysis of the excised DGGE bands revealed the genera of Niabella, Sphingobium, Bellilinea and Burkholderiales (see Table 2). As has been reported, the genus of Bellilinea comprises obligately anaerobic bacteria (33), whereas the others comprise aerobic or facultative aerobic bacteria (34e36). Most were heterotrophic bacteria, even though inorganic wastewater was fed to the reactor. In situ probing of anammox activity within granules To investigate the in situ microbial activity within granules, four microelectrodes were applied to measure the microprofiles of pH, NHþ 4 , NO2 and NO3 . The medium used for the measurement of anammox activity contained nitrite and ammonium at a moler
FIG. 4. Steady-state microprofiles of pH, NHþ 4 , NO2 and NO3 within the granules at a sufficient substrate concentration. (AeC) Ammonium concentrations of (A) 100, (B) 500 and (C) 1000 mmol L1. Net volumetric production rates of NH4þ, NO2 and NO3 within the granules. (DeF) Ammonium concentrations of (D) 100, (E) 500 and (F) 1000 mmol L1.
Please cite this article in press as: Lv, Y., et al., In situ probing of microbial activity within anammox granular biomass with microelectrodes, J. Biosci. Bioeng., (2015), http://dx.doi.org/10.1016/j.jbiosc.2015.08.015
VOL. xx, 2015 were obtained by fitting the concentration profile using the software Sensor Trace PRO. The net volumetric consumption or production rates were calculated and are shown in Fig. 4DeF. Negative and positive values indicate production and consumption, respectively. Throughout the external diffusive boundary layer (DBL), internal diffusion together with the biological reaction resulted in a curved profile. On the other side, the nearly vertical part of the profile (perpendicular to the granule surface) belonged to the bulk liquid phase. Therefore, the interface between the granule and the bulk solution was located in Fig. 4. In addition, due to the cross-flow pattern over the granule, the thickness of the DBL was almost negligible, which was in agreement with observations of Zhou et al. (37). The trends of the microprofiles within the granules were similar at different substrate concentrations (Fig. 4AeC). With increasing depth, the concentrations of nitrite and ammonium decreased simultaneously, and both depleted at around 2000 mm, while the nitrate concentration and pH value increased slightly. The concentration microprofiles coincide with the observation of anammox biofilm (6). The net volumetric rates within the granular biomass reflect substrate diffusion, consumption or production. Under different substrate concentrations, the trends of the net volumetric consumption rates within granules were similar (Fig. 4DeF). Net volumetric consumption rates of both nitrite and ammonium were observed in the upper layer (<1500 mm) of the granules, and the consumption rate of nitrite was a little higher than that of ammonium. This revealed an anammox reaction. The obtained consumption rate of the most active layer was limited to the surface layer of 200e400 mm. With increasing depth from 500 mm, anammox activity decreased. The in situ anammox activity increased with increasing substrate concentration. Maximum net volumetric consumption rates of ammonium of 8.7, 39.6 and 83.3 mmol cm3 h1 were obtained at ammonium concentrations of 100, 500 and 1000 mmol L1 in the medium, respectively. The active zone of anammox extended from around 1500 mm (Fig. 4D, E) to 2000 mm (Fig. 4F) when the ammonium concentration increased from 100 to 1000 mmol L1. Some work has been performed to describe in situ anammox activity using microelectrodes (6,27,38). The anammox active zone and activity in the present study differ from those of different bacterial aggregates. Cho et al. (27) identified the active zone of anammox in the upper layer of 800 mm, similar to what had been
IN SITU ACTIVITY WITHIN ANAMMOX GRANULE
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reported previously (6,38). However, the active zone observed in this study was much deeper than that observed in other studies, and therefore, a high ammonium consumption rate of 83.3 mmol cm3 h1 was obtained. In situ probing of denitrification activity within granules To identify the denitrification activity within anammox granules, two ammonium-limited substrates were used for microprofile measurements. One contained nitrite and ammonium at a ratio of 2:1. The other contained only nitrite at a concentration of 600 mmol L1. When the moler ratio of nitrite to ammonium was 2:1, both ammonia and nitrite concentrations decreased in the outer layer (0e1000 mm). In the inner layer (1100e2500 mm), a decrease in the nitrite concentration was also observed under the ammoniumlimited condition (Fig. 5A). The spatial distributions of ammonium and nitrite rates indicate that high anammox activity was restricted to the upper 1000 mm, with the maximum ammonium consumption rate being 9.2 mmol cm3 h1 at 200 mm (Fig. 5B). In the inner layer (>1000 mm), where almost no ammonium was consumed, a higher nitrite consumption rate was observed. This revealed a denitrification reaction. The pH value first increased from 8.09 to 8.31 at 1300 mm and then decreased to around 8.29, thus indicating the microbial reaction. When the substrate only contained nitrite (at a concentration of approximately 600 mmol L1), in the upper layer of 1500 mm, there was an obvious decrease in the nitrite concentration (Fig. 5C) similar to that in the investigation of Xiao et al. (14). The consumption rate of nitrite revealed denitrification (Fig. 5D). With increasing depth, denitrification activity first increased and then decreased. The most active layer was limited to 700e1000 mm of the inner layer, and a maximum nitrite consumption rate of 27.5 mmol cm3 h1 was obtained at 800 mm. Multiple bacterial populations usually coexist in an anammox system (4,6,39), and microelectrodes have been identified as the most indispensable tools for in situ analysis of microbial activity. In this study, inorganic synthetic wastewater was fed to a reactor, yet some heterotrophic bacteria were detected by PCR and DGGE analysis. And some of them were heterotrophic bacteria, might be capable using nitrite or nitrate as electron acceptor. So that denitrification activity was detected in situ by microelectrodes. This indicated the two kinds of bacteria tended to coexist, because the
FIG. 5. Steady-state microprofiles of pH, NH4þ, NO2 and NO3 within the granules under ammonium-limited conditions. (A corresponds to nitrite and ammonium at a moler ratio of 2:1, C corresponds to nitrite concentration of 600 mmol L1). Net volumetric production rates of NHþ 4 , NO2 and NO3 within the granules (B corresponds to nitrite and ammonium at a moler ratio of 2:1, D corresponds to nitrite concentration of 600 mmol L1).
Please cite this article in press as: Lv, Y., et al., In situ probing of microbial activity within anammox granular biomass with microelectrodes, J. Biosci. Bioeng., (2015), http://dx.doi.org/10.1016/j.jbiosc.2015.08.015
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by-product nitrate of anammox offered the substrate for denitrification bacteria and the organic compounds were derived from biomass decay (6). Therefore, when organic concentration increased, excess growth of denitrification bacteria in anammox system was observed (8). In this study, the in situ denitrification activity was much lower than that of anammox due to the lack of organic matter. The microscopic results obtained in this study reveal that (i) under an ammonium-limited condition, excess nitrite can be partially removed from the inner layer of granules and (ii) a small amount of organic matter might be helpful in evaluating the TN removal efficiency owing to the coaction of anammox and denitrification. Therefore, the microscopic results will allow future optimization of the anammox performance. In conclusion, in this study besides biofilm, granules formed in an AnRBC reactor. Anammox and denitrification activities were measured in situ within the granules using microelectrodes. At a sufficient substrate concentration, an anammox reaction was observed in the upper layer, and the most active zone was in the surface layer of 200e400 mm. Under an ammonium-limited condition, denitrification activity was observed in the inner layer and the most active zone was limited to depths of 700e1000 mm. This study revealed that denitrification bacteria coexisted with anammox bacteria even though inorganic wastewater was fed to the reactor. ACKNOWLEDGMENTS This work was supported by the National Natural Science Foundation of China (No. 51108367), Specialized Research Fund for the Doctoral Program of Higher Education (No. 20116120120009), Natural Science Foundation of Shaan Xi Province (No. 2014JQ7243), Provincial Key Laboratory Projects of Environmental Engineering of Education Department of Shaanxi Province (No. 11JS056) and Innovative Research Team of Xi’an University of Architecture and Technology. References 1. Jetten, M. S., Strous, M., van de Pas-Schoonen, K. T., Schalk, J., van Dongen, U. G., van de Graaf, A. A., Logemann, S., Muyzer, G., van Loosdrecht, M. C., and Kuenen, J. G.: The anaerobic oxidation of ammonium, FEMS Microbiol. Rev., 22, 421e437 (1998). 2. Rodriguez-Garcia, G., Frison, N., Vázquez-Padín, J. R., Hospido, A., Garrido, J. M., Fatone, F., Bolzonella, D., Moreira, M. T., and Feijoo, G.: Life cycle assessment of nutrient removal technologies for the treatment of anaerobic digestion supernatant and its integration in a wastewater treatment plant, Sci. Total Environ., 490, 871e879 (2014). 3. Strous, M., Heijnen, J. J., Kuenen, J. G., and Jetten, M. S. M.: The sequencing batch reactor as a powerful tool for the study of slowly growing anaerobic ammonium-oxidizing microorganisms, Appl. Microbiol. Biotechnol., 50, 589e596 (1998). 4. Kumar, M. and Lin, J. G.: Co-existence of anammox and denitrification for simultaneous nitrogen and carbon removaldStrategies and issues, J. Hazard. Mater., 178, 1e9 (2010). 5. Kim, W., Inge, D. B., and Willy, V.: Oxygen-limited autotrophic nitrificationdenitrification (OLAND) in a rotating biological contactor treating highsalinity wastewater, Water Res., 39, 4512e4520 (2005). 6. Kindaichi, T., Tsushima, I., Ogasawara, Y., Shimokawa, M., Ozaki, N., Satoh, H., and Okabe, S.: In situ activity and spatial organization of anaerobic ammonium-oxidizing (anammox) bacteria in biofilms, Appl. Environ. Microb., 73, 4931e4939 (2007). 7. Rikmann, E., Zekker, I., Tomingas, M., Vabamäe, P., Kroon, K., Saluste, A., Tenno, T., Menert, A., Loorits, L., Rubin, S. S. C. D., and Tenno, T.: Comparison of sulfate-reducing and conventional Anammox upflow anaerobic sludge blanket reactors, J. Biosci. Bioeng., 118, 61e63 (2014). 8. Du, R., Peng, Y., Cao, S., Wu, C., Weng, D., Wang, S., and He, J.: Advanced nitrogen removal with simultaneous Anammox and denitrification in sequencing batch reactor, Bioresour. Technol., 162, 316e322 (2014). 9. Takekawa, M., Park, G., Soda, S., and Ike, M.: Simultaneous anammox and denitrification (SAD) process in sequencing batch reactors, Bioresour. Technol., 174, 159e166 (2014).
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Cho, S., Takahashi, Y., Fujii, N., Yamada, Y., Satoh, H., and Okabe, S.: Nitrogen removal performance and microbial community analysis of an anaerobic up-flow granular bed anammox reactor, Chemosphere, 78, 1129e1135 (2010). 28. Ni, S. Q., Sung, S., Yue, Q. Y., and Gao, B. Y.: Substrate removal evaluation of granular anammox process in a pilot-scale upflow anaerobic sludge blanket reactor, Ecol. Eng., 38, 30e36 (2012). 29. Xing, B. S., Guo, Q., Zhang, Z. Z., Zhang, J., Wang, H. Z., and Jin, R. C.: Optimization of process performance in a granule-based anaerobic ammonium oxidation (anammox) upflow anaerobic sludge blanket (UASB) reactor, Bioresour. Technol., 170, 404e412 (2014). 30. Fernández, I., Vázquez-Padín, J. R., Mosquera-Corral, A., Campos, J. L., and Méndez, R.: Biofilm and granular systems to improve Anammox biomass retention, Biochem. Eng. J., 42, 308e313 (2008). 31. Rosman, N. H., Nor Anuar, A., Chelliapan, S., Md Din, M. F., and Ujang, Z.: Characteristics and performance of aerobic granular sludge treating rubber wastewater at different hydraulic retention time, Bioresour. Technol., 161, 155e161 (2014). 32. Li, Z., Xu, X., Shao, B., Zhang, S., and Yang, F.: Anammox granules formation and performance in a submerged anaerobic membrane bioreactor, Chem. Eng. J., 254, 9e16 (2014). 33. Yamada, T., Imachi, H., Ohashi, A., Harada, H., Hanada, S., Kamagata, Y., and Sekiguchi, Y.: Bellilinea caldifistulae gen. nov., sp. nov. and Longilinea arvoryzae gen. nov., sp. nov., strictly anaerobic, filamentous bacteria of the phylum
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VOL. xx, 2015 Chloroflexi isolated from methanogenic propionate-degrading consortia, Int. J. Syst. Evol. Microbiol., 57, 2299e2306 (2007). 34. Niemi, R. M., Heiskanen, I., Heine, R., and Rapala, J.: Previously uncultured bProteobacteria dominate in biologically active granular activated carbon (BAC) filters, Water Res., 43, 5075e5086 (2009). 35. Nagata, Y., Natsui, S., Endo, R., Ohtsubo, Y., Ichikawa, N., Ankai, A., Oguchi, A., Fukui, S., Fujita, N., and Tsuda, M.: Genomic organization and genomic structural rearrangements of Sphingobium japonicum UT26, an archetypal g-hexachlorocyclohexane-degrading bacterium, Enzyme Microb. Technol., 49, 499e508 (2011). 36. Kämpfer, P., Lodders, N., and Falsen, E.: Hydrotalea flava gen. nov., sp. nov., a new member of the phylum Bacteroidetes and allocation of the genera Chitinophaga, Sediminibacterium, Lacibacter, Flavihumibacter, Flavisolibacter,
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Niabella, Niastella, Segetibacter, Parasegetibacter, Terrimonas, Ferruginibacter, Filimonas and Hydrotalea to the family Chitinophagaceae fam. nov., Int. J. Syst. Bacteriol., 61, 518e523 (2011). 37. Zhou, X.-H., Shi, H.-C., Cai, Q., He, M., and Wu, Y.-X.: Function of self-forming dynamic membrane and biokinetic parameters’ determination by microelectrode, Water Res., 42, 2369e2376 (2008). 38. Tsushima, I., Ogasawara, Y., Kindaichi, T., Satoh, H., and Okabe, S.: Development of high-rate anaerobic ammonium-oxidizing (anammox) biofilm reactors, Water Res., 41, 1623e1634 (2007). 39. Truu, M., Juhanson, J., and Truu, J.: Microbial biomass, activity and community composition in constructed wetlands, Sci. Total Environ., 407, 3958e3971 (2009).
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In situ probing of microbial activity within anammox granular biomass with microelectrodes Yongtao Lv,1 Kai Ju,1 Lei Wang,1, * Ting Sun,1 Rui Miao,1 Xudong Wang,1 Fan Wei,1 and Siqing Xia2 School of Environmental and Municipal Engineering, Xi’an University of Architecture and Technology, Xi’an 710055, China1 and College of Environmental Science and Engineering, Tongji University, Shanghai 200092, China2 Received 23 April 2015; accepted 29 August 2015 Available online xxx
An anaerobic rotating biological contactor was fed with inorganic synthetic wastewater for anammox. Besides bioL L film, granular biomass with average diameter of approximately 5 mm formed. NHD 4 , NO2 , NO3 and pH microelectrodes were used to probe microbial activity in situ within the granules. At a sufficient substrate concentration, the anammox reaction was observed in the upper layer of granules, and the most active zone was found to be in the surface of 200e400 mm. The in situ anammox activity increased with increasing substrate concentration, and a maximum ammonium consumption rate of 83.3 mmol cmL3 hL1 was obtained at an ammonium concentration of 1000 mmol LL1. Under an ammonium-limited condition, denitrification activity was observed in the inner layer, and the most active zone was limited to 700e1000 mm. This study revealed that denitrification bacteria coexisted with anammox bacteria within inorganic anammox granules. Ó 2015, The Society for Biotechnology, Japan. All rights reserved. [Key words: Anammox; Denitrification; In situ microbial activity; Active zone; Microelectrodes]
Nitrogen removal from wastewater has become an important part of the overall treatment of wastewater because of the significant effect of nitrogen compounds on the groundwater environment (1). However, some wastewater rich in NHþ 4 but poor in biodegradable organic carbon (for example, digested sludge supernatant and landfill leachate) is difficult to treat with the application of a conventional nitrificationedenitrification process. In such cases, anaerobic ammonium oxidation (anammox) has been found to be an alternative process for ammonium removal in the absence of organic matter and oxygen (2). In this process, autotrophic anammox bacteria oxidize ammonium to N2 using nitrite (NO 2 ) as an electron acceptor under strict anoxic conditions. The stoichiometry of this reaction proposed by Strous et al. (3) is shown in Eq. 1. þ NHþ 4 þ 1.32NO2 þ 0.066HCO3 þ 0.13H / 1.02N2 þ 0.26NO3 þ 2.03H2O þ 0.066CH2O0.5N0.15 (1)
To supply nitrite as an electron acceptor and realize an autotrophic nitrogen removal process, anammox is often combined with nitritation. The combined process can reduce operation costs by nearly 40% and is considered an economic and promising way of treating ammonium-rich wastewater (4). Anammox bacteria are mostly anaerobic autotrophs and thus sensitive to concentrations of dissolved oxygen (DO) and organic matter (5,6). However, wastewater is a complex ecosystem;
* Corresponding author. Tel./fax: þ86 29 82202729. E-mail address: [email protected] (L. Wang).
therefore, multiple bacteria usually coexist in an anammox system. Kumar and Lin (4) demonstrated that anammox and denitrification were responsible for the removal of nitrogen from low-C/N wastewater. Kindaichi et al. (6) found that ammonium-oxidizing bacteria (AOB), nitrite-oxidizing bacteria (NOB) and anammox bacteria coexisted in an anammox system (6). The biomass populations and nitrogen removal performance have been well studied by fluorescence in situ hybridization analysis and stoichiometry analysis (5,7e9). However, there is limited knowledge about substrate diffusion and consumption inside bacterial aggregates, which is essential for clarifying the microbial reaction and improving process performance. Microelectrodes, which are needle-shaped devices with sensitive tips, have been demonstrated to be the most suitable tools for the in situ profiling of substrate concentrations within bacterial aggregates (10e12). Studies have investigated the spatial distributions of microbial activity. Gieseke et al. (11) studied substrate transport through biofilm and determined the size of an active zone. Li and Bishop (12) investigated the microprofiles of substrate concentrations within activated sludge flocs and offered an optimization strategy. Satoh et al. (13) and Xiao et al. (14) studied variations in nitrification and denitrification activity within biofilm under different conditions. However, only a few studies have investigated in situ activities of multiple bacteria in anammox aggregates using microelectrodes (6,15,16). A better understanding of the in situ microbial activity will allow further optimization of the reactor. In our previous study (17), an anaerobic rotating biological contactor (AnRBC) was used for anammox and removed more than 90% of total nitrogen (TN) from influent. Besides biofilm attaching to the surface of the discs, we observed the formation
1389-1723/$ e see front matter Ó 2015, The Society for Biotechnology, Japan. All rights reserved. http://dx.doi.org/10.1016/j.jbiosc.2015.08.015
Please cite this article in press as: Lv, Y., et al., In situ probing of microbial activity within anammox granular biomass with microelectrodes, J. Biosci. Bioeng., (2015), http://dx.doi.org/10.1016/j.jbiosc.2015.08.015
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TABLE 1. In situ microbial activity experiments. Concentration (mmol L
No.
1 2 3 4 5
MATERIALS AND METHODS
1
)
NHþ 4
NO 2
NO 3
100 500 1000 100 0
120 600 1200 200 600
0 0 0 0 0
of granules. The objectives of the present study were to identify the spatial distribution of microbial activity within the granular biomass and to investigate the effect of the substrate concentration on microbial activity. To this end, the microbial community present was firstly investigated using polymerase chain reaction (PCR) and denatured gradient gel electrophoresis (DGGE) analysis. And then, pH, NHþ 4 , NO2 and NO3 microelectrodes were used to measure microprofiles under different substrate concentrations. All results obtained from a microscopic view provide valuable information for better understanding the reactor performance.
Experimental set-up Anammox performance was investigated using an AnRBC with a net liquid capacity of 6.2 L that has been operated for more than 7 years (17). The AnRBC has 13 polyvinyl chloride discs, with a total surface area of 0.32 m2 and 87% submersion of the disc surface. The rotation speed of the discs was controlled at 1.3e1.5 r min1. The hydraulic retention time was kept at 1 d. The reactor was maintained at 40 1 C using a water-jacket. Synthetic wastewater The synthetic wastewater used in this study mainly contained NaHCO3, NH4Cl, NaNO2 and trace elements described by van de Graaf et al. (18). The concentrations of NHþ 4 -N and NO2 -N were kept in the range 200e280 mg L1. The pH value of influent was maintained between 7.5 and 8.0. Chemical analysis The concentration of NHþ 4 -N was determined according to standard methods of APHA (19). NO 3 -N and NO2 -N were determined using an ion chromatograph (Metrohm 761, Metrohm, Switzerland). The DO level was measured with a digital portable HI9147 Oxy-check (Hanna, Romania). DNA extraction, PCR-DGGE, cloning, and sequencing The total DNA was extracted using a bacterial genomic mini extraction kit (Sangon, China). To identify the genus of AnAOB, specific primers of AMX818F and AMX1066R were used for the PCR amplification (20). To investigate the community structure, universal primers F357 (with GC clamp) and R518 were used for the PCR amplification (21). For DGGE analysis, the PCR products were purified using a gel midi purification kit (Sangon, China) and
FIG. 1. Time course of the nitrogen removal performance of the AnRBC.
FIG. 2. Morphology (A) and Scanning electron microscopy images (B) of the ANAMMOX granules.
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separated using DGGE in 8% (wt/vol) polyacrylamide gels containing a linear gradient of 30%e60% denaturant employing a DCode Universal Mutation Detection System (Bio-Rad, USA). Prominent DGGE bands in the gel were excised and dissolved in 100 mL of 1 TE buffer at 4 C overnight. A 1 mL volume of TE solution as a template was reamplified with the primer pairs (without the GC clamp) using the same method as described previously, followed by purification using a purification kit (Sangon, China) and cloning in a pMD19-T plasmid vector system (Takara, Japan). Sequencing was performed using an ABI 3730 DNA sequencer offered by a commercial service (Sangon, China). All the sequences obtained were retrieved from the GenBank database by employing the BLAST alignment tool at the National Center for Biotechnology Information (NCBI). Microsensor measurements Microprofiles of pH and the NHþ 4 , NO2 and NO 3 concentrations within the bacterial aggregates were obtained using micro electrodes. Microelectrodes for measuring pH, NHþ 4 , NO2 and NO3 with tip diameters of approximately 15 mm were manufactured in our laboratory and their performances were confirmed to be stable (22). Before making microprofile measurements, the microelectrodes were prepared, calibrated and operated as described by Satoh et al. (23) and Okabe et al. (24). Although the AnRBC was a biofilm reactor, granular biomass with an average diameter of around 5 mm also formed in our study. Granules were sampled from the inlet of the reactor, and a chamber was used for microsensor measurements (10). After sampling, a single granule was placed gently just above the nylon net in the chamber, and then fixed by two needles. To obtain steady-state profiles, the granule was left for at least 1 h in preincubation before profile measurements were taken. Measurements were made at least three times with each microelectrode. To investigate the in situ activity of anammox bacteria and other bacteria, substrates with different concentrations were used to measure microprofiles (Table 1). Different granules were used at three levels of ammonium concentrations. To remove the batch-to-batch variation, granules with diameter of 5 mm were selected for microprofile measurements. The DO concentration of this medium was kept below 0.05 mg L1 through continuous purging with N2.
Fluxes and rates calculations The following basic assumptions will be made for fluxes and rates calculations: (i) No substrate consumption or production occur in the bulk liquid; (ii) the granules are spherical in shape and uniform in size; (iii) only radial diffusion transport is considered and is described by Fick’s law. Firstly, simulated curves were obtained by fitting the concentration profile using the software Sensor Trace PRO. Secondly, diffusive fluxes are calculated according to Fick’s first law (25): Jn ¼ Ds
dCn C Cn1 ¼ Ds nþ1 drn rnþ1 rn1
(2)
where Jn is the flux at point n (mmol m2 s1), Ds is the effective diffusion coefficient (m2 s1), dCn/drn is the concentration gradient at point n (mmol m3 m1), C is the simulated substrate concentration (mmol m3), and r is the distance from the granule center (m). Values of 1.23 109, 1.1 109 and 1.5 109 m2 s1 were used for diffusion coefficients of nitrite, ammonium and nitrate, respectively (12)). Finally, the net volumetric consumption or production rates (Rn, mmol m3 s1) of NHþ 4 , NO2 and NO3 in each layer n were calculated from the steady-state concentration profiles using Fick’s second law of diffusion (26): Rn ¼
2 2 4prnþ1 Jnþ1 4prn1 Jn1 4 pr 3 3 nþ1
4 pr 3 3 n1
¼ 3
2 2 rnþ1 Jnþ1 rn1 Jn1 3 rnþ1
! (3)
3 rn1
RESULTS AND DISCUSSION Performance of the AnRBC The laboratory-scale AnRBC has been operated in anammox for more than 7 years. During the period of this study, inorganic synthetic wastewater was fed to the reactor. The nitrogen removal performance is shown in Fig. 1.
TABLE 2. The microbial community from the AnRBC. Primers AMX818F/AMX1066R F357/R518
FIG. 3. DGGE analysis of total bacteria in the AnRBC. Detailed information on the bands 1e5 is presented in Table 2.
Closest relative
Identity (%)
Candidatus Kuenenia stuttgartiensis Niabella sp. Sphingobium xenophagum Bellilinea sp. Bellilinea sp. Burkholderiales
100 100 100 96.3 92 100
Band e 1 2 3 4 5
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During 100 days of operation, the influent TN concentration was 491.2 23.9 mg L1, the AnRBC removed 91.4% 1.6% of TN, and 0.5 kg m3 d1 of maximum TN removal volume load was obtained. Nitrite and ammonium were removed simultaneously, and the effluent concentrations of nitrite and ammonia were 2.6 2.6 and 2.3 2.8 mg L1, respectively. In contrast, the nitrate concentration increased slightly, from 29.9 5.9 mg L1 in influent to 37.2 6.1 mg L1 in effluent. Nitrate was also detected in influent, it might be mainly caused by the insufficient reagent purity which used in this experiment. At an influent nitrite concentration of 200e280 mg L1, no inhibition of anammox biomass activity was observed in our system, which was in accordance with the result of Cho et al. (27). These results demonstrate that the stratified granules or biofilm biomass tolerated a higher NO 2 concentration than did the homogenized biomass owing to limited diffusion. Because of the complex wastewater quality, multiple bacteria usually coexist in an anammox reactor, e.g., AOB, NOB and denitrifying bacteria. In the present anammox system, the moler ratio of consumed nitrite to ammonium was 1.1 0.1, and the moler ratio of produced nitrate to consumed ammonium was 0.03 0.02. Both values were lower than the ratios in Eq. 1, which demonstrated the presence of another nitrogen removal pathway.
J. BIOSCI. BIOENG., ratio of 1.2:1. Three media having gradient ammonium concentrations of 100, 500 and 1000 mmol L1 were used. The concentration profiles were measured at least three times, and the average profiles are presented in Fig. 4AeC. The simulated curves
Sludge morphology and structure of the microbial community Besides biofilm attaching to the surface of the discs, granular biomass formed in the reactor, especially at the inlet of the reactor. The average diameter of the granules was approximately 5 mm (Fig. 2A), and aggregates of spherical bacteria were observed on the surfaces of the granules with a scanning electron microscope (Fig. 2B). Anammox bacteria are sensitive to environmental conditions (e.g., substrate concentration, DO, pH), and they thus likely grow into stratified granules or biofilm. Anammox granular biomass has been reported in many reactors, such as the upflow anaerobic sludge bed reactor (28,29), upflow granular reactor (27), sequencing batch reactor (8,30,31) and membrane reactor (32). However, granule formation in an AnRBC biofilm reactor has not yet been reported. In our previous study (17), anammox bacteria accounted for approximately 80% of the total biomass, both in granules and biofilms. Therefore, in this study, the AnRBC is a hybrid system in which both attached and suspended sludge coexist. Further studies are needed to clarify the contributions of the two morphological structures of sludge to the nitrogen removal efficiency). Around 7 years and 2 months after the start-up of anammox, the microbial community was investigated using PCR and DGGE analyses. Using the primers AMX818F and AMX1066R, PCR products of 16SrDNA with full length of 241 bp were obtained. Analysis of the DNA sequence revealed that the AnAOB belonged to the genus of Candidatus Kuenenia stuttgartiensis. Using universal primers F357 (with a GC clamp) and R518, five discrete DGGE bands were excised (see Fig. 3), and the DNA was eluted and sequenced. The DNA sequence analysis of the excised DGGE bands revealed the genera of Niabella, Sphingobium, Bellilinea and Burkholderiales (see Table 2). As has been reported, the genus of Bellilinea comprises obligately anaerobic bacteria (33), whereas the others comprise aerobic or facultative aerobic bacteria (34e36). Most were heterotrophic bacteria, even though inorganic wastewater was fed to the reactor. In situ probing of anammox activity within granules To investigate the in situ microbial activity within granules, four microelectrodes were applied to measure the microprofiles of pH, NHþ 4 , NO2 and NO3 . The medium used for the measurement of anammox activity contained nitrite and ammonium at a moler
FIG. 4. Steady-state microprofiles of pH, NHþ 4 , NO2 and NO3 within the granules at a sufficient substrate concentration. (AeC) Ammonium concentrations of (A) 100, (B) 500 and (C) 1000 mmol L1. Net volumetric production rates of NH4þ, NO2 and NO3 within the granules. (DeF) Ammonium concentrations of (D) 100, (E) 500 and (F) 1000 mmol L1.
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VOL. xx, 2015 were obtained by fitting the concentration profile using the software Sensor Trace PRO. The net volumetric consumption or production rates were calculated and are shown in Fig. 4DeF. Negative and positive values indicate production and consumption, respectively. Throughout the external diffusive boundary layer (DBL), internal diffusion together with the biological reaction resulted in a curved profile. On the other side, the nearly vertical part of the profile (perpendicular to the granule surface) belonged to the bulk liquid phase. Therefore, the interface between the granule and the bulk solution was located in Fig. 4. In addition, due to the cross-flow pattern over the granule, the thickness of the DBL was almost negligible, which was in agreement with observations of Zhou et al. (37). The trends of the microprofiles within the granules were similar at different substrate concentrations (Fig. 4AeC). With increasing depth, the concentrations of nitrite and ammonium decreased simultaneously, and both depleted at around 2000 mm, while the nitrate concentration and pH value increased slightly. The concentration microprofiles coincide with the observation of anammox biofilm (6). The net volumetric rates within the granular biomass reflect substrate diffusion, consumption or production. Under different substrate concentrations, the trends of the net volumetric consumption rates within granules were similar (Fig. 4DeF). Net volumetric consumption rates of both nitrite and ammonium were observed in the upper layer (<1500 mm) of the granules, and the consumption rate of nitrite was a little higher than that of ammonium. This revealed an anammox reaction. The obtained consumption rate of the most active layer was limited to the surface layer of 200e400 mm. With increasing depth from 500 mm, anammox activity decreased. The in situ anammox activity increased with increasing substrate concentration. Maximum net volumetric consumption rates of ammonium of 8.7, 39.6 and 83.3 mmol cm3 h1 were obtained at ammonium concentrations of 100, 500 and 1000 mmol L1 in the medium, respectively. The active zone of anammox extended from around 1500 mm (Fig. 4D, E) to 2000 mm (Fig. 4F) when the ammonium concentration increased from 100 to 1000 mmol L1. Some work has been performed to describe in situ anammox activity using microelectrodes (6,27,38). The anammox active zone and activity in the present study differ from those of different bacterial aggregates. Cho et al. (27) identified the active zone of anammox in the upper layer of 800 mm, similar to what had been
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reported previously (6,38). However, the active zone observed in this study was much deeper than that observed in other studies, and therefore, a high ammonium consumption rate of 83.3 mmol cm3 h1 was obtained. In situ probing of denitrification activity within granules To identify the denitrification activity within anammox granules, two ammonium-limited substrates were used for microprofile measurements. One contained nitrite and ammonium at a ratio of 2:1. The other contained only nitrite at a concentration of 600 mmol L1. When the moler ratio of nitrite to ammonium was 2:1, both ammonia and nitrite concentrations decreased in the outer layer (0e1000 mm). In the inner layer (1100e2500 mm), a decrease in the nitrite concentration was also observed under the ammoniumlimited condition (Fig. 5A). The spatial distributions of ammonium and nitrite rates indicate that high anammox activity was restricted to the upper 1000 mm, with the maximum ammonium consumption rate being 9.2 mmol cm3 h1 at 200 mm (Fig. 5B). In the inner layer (>1000 mm), where almost no ammonium was consumed, a higher nitrite consumption rate was observed. This revealed a denitrification reaction. The pH value first increased from 8.09 to 8.31 at 1300 mm and then decreased to around 8.29, thus indicating the microbial reaction. When the substrate only contained nitrite (at a concentration of approximately 600 mmol L1), in the upper layer of 1500 mm, there was an obvious decrease in the nitrite concentration (Fig. 5C) similar to that in the investigation of Xiao et al. (14). The consumption rate of nitrite revealed denitrification (Fig. 5D). With increasing depth, denitrification activity first increased and then decreased. The most active layer was limited to 700e1000 mm of the inner layer, and a maximum nitrite consumption rate of 27.5 mmol cm3 h1 was obtained at 800 mm. Multiple bacterial populations usually coexist in an anammox system (4,6,39), and microelectrodes have been identified as the most indispensable tools for in situ analysis of microbial activity. In this study, inorganic synthetic wastewater was fed to a reactor, yet some heterotrophic bacteria were detected by PCR and DGGE analysis. And some of them were heterotrophic bacteria, might be capable using nitrite or nitrate as electron acceptor. So that denitrification activity was detected in situ by microelectrodes. This indicated the two kinds of bacteria tended to coexist, because the
FIG. 5. Steady-state microprofiles of pH, NH4þ, NO2 and NO3 within the granules under ammonium-limited conditions. (A corresponds to nitrite and ammonium at a moler ratio of 2:1, C corresponds to nitrite concentration of 600 mmol L1). Net volumetric production rates of NHþ 4 , NO2 and NO3 within the granules (B corresponds to nitrite and ammonium at a moler ratio of 2:1, D corresponds to nitrite concentration of 600 mmol L1).
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by-product nitrate of anammox offered the substrate for denitrification bacteria and the organic compounds were derived from biomass decay (6). Therefore, when organic concentration increased, excess growth of denitrification bacteria in anammox system was observed (8). In this study, the in situ denitrification activity was much lower than that of anammox due to the lack of organic matter. The microscopic results obtained in this study reveal that (i) under an ammonium-limited condition, excess nitrite can be partially removed from the inner layer of granules and (ii) a small amount of organic matter might be helpful in evaluating the TN removal efficiency owing to the coaction of anammox and denitrification. Therefore, the microscopic results will allow future optimization of the anammox performance. In conclusion, in this study besides biofilm, granules formed in an AnRBC reactor. Anammox and denitrification activities were measured in situ within the granules using microelectrodes. At a sufficient substrate concentration, an anammox reaction was observed in the upper layer, and the most active zone was in the surface layer of 200e400 mm. Under an ammonium-limited condition, denitrification activity was observed in the inner layer and the most active zone was limited to depths of 700e1000 mm. This study revealed that denitrification bacteria coexisted with anammox bacteria even though inorganic wastewater was fed to the reactor. ACKNOWLEDGMENTS This work was supported by the National Natural Science Foundation of China (No. 51108367), Specialized Research Fund for the Doctoral Program of Higher Education (No. 20116120120009), Natural Science Foundation of Shaan Xi Province (No. 2014JQ7243), Provincial Key Laboratory Projects of Environmental Engineering of Education Department of Shaanxi Province (No. 11JS056) and Innovative Research Team of Xi’an University of Architecture and Technology. References 1. Jetten, M. S., Strous, M., van de Pas-Schoonen, K. T., Schalk, J., van Dongen, U. G., van de Graaf, A. A., Logemann, S., Muyzer, G., van Loosdrecht, M. C., and Kuenen, J. G.: The anaerobic oxidation of ammonium, FEMS Microbiol. Rev., 22, 421e437 (1998). 2. Rodriguez-Garcia, G., Frison, N., Vázquez-Padín, J. R., Hospido, A., Garrido, J. M., Fatone, F., Bolzonella, D., Moreira, M. T., and Feijoo, G.: Life cycle assessment of nutrient removal technologies for the treatment of anaerobic digestion supernatant and its integration in a wastewater treatment plant, Sci. Total Environ., 490, 871e879 (2014). 3. Strous, M., Heijnen, J. J., Kuenen, J. G., and Jetten, M. S. M.: The sequencing batch reactor as a powerful tool for the study of slowly growing anaerobic ammonium-oxidizing microorganisms, Appl. Microbiol. Biotechnol., 50, 589e596 (1998). 4. Kumar, M. and Lin, J. G.: Co-existence of anammox and denitrification for simultaneous nitrogen and carbon removaldStrategies and issues, J. Hazard. Mater., 178, 1e9 (2010). 5. Kim, W., Inge, D. B., and Willy, V.: Oxygen-limited autotrophic nitrificationdenitrification (OLAND) in a rotating biological contactor treating highsalinity wastewater, Water Res., 39, 4512e4520 (2005). 6. Kindaichi, T., Tsushima, I., Ogasawara, Y., Shimokawa, M., Ozaki, N., Satoh, H., and Okabe, S.: In situ activity and spatial organization of anaerobic ammonium-oxidizing (anammox) bacteria in biofilms, Appl. Environ. Microb., 73, 4931e4939 (2007). 7. Rikmann, E., Zekker, I., Tomingas, M., Vabamäe, P., Kroon, K., Saluste, A., Tenno, T., Menert, A., Loorits, L., Rubin, S. S. C. D., and Tenno, T.: Comparison of sulfate-reducing and conventional Anammox upflow anaerobic sludge blanket reactors, J. Biosci. Bioeng., 118, 61e63 (2014). 8. Du, R., Peng, Y., Cao, S., Wu, C., Weng, D., Wang, S., and He, J.: Advanced nitrogen removal with simultaneous Anammox and denitrification in sequencing batch reactor, Bioresour. Technol., 162, 316e322 (2014). 9. Takekawa, M., Park, G., Soda, S., and Ike, M.: Simultaneous anammox and denitrification (SAD) process in sequencing batch reactors, Bioresour. Technol., 174, 159e166 (2014).
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Please cite this article in press as: Lv, Y., et al., In situ probing of microbial activity within anammox granular biomass with microelectrodes, J. Biosci. Bioeng., (2015), http://dx.doi.org/10.1016/j.jbiosc.2015.08.015