Hori, T.

Hosomi, M.

Kinh, C. T.

Riya, S.

Smets, Barth F.

Suenaga, T.

Terada, A.

Online Research Database In Technology

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Downloaded from orbit.dtu.dk on: Dec 18, 2017Spatial distribution of microbial community and N2O depth profiles in counter- and co-diffusion biofilms functioning simultaneous nitrification and denitrificationTerada, A.; Kinh, C. T.; Suenaga, T.; Hori, T.; Riya, S.; Smets, Barth F.; Hosomi, M.Published in:Microbial ecology and water engineering &amp; biofilms specialist groups (MEWE2016)Publication date:2016Document VersionPublisher's PDF, also known as Version of recordLink back to DTU OrbitCitation (APA):Terada, A., Kinh, C. T., Suenaga, T., Hori, T., Riya, S., Smets, B. F., & Hosomi, M. (2016). Spatial distribution ofmicrobial community and N2O depth profiles in counter- and co- diffusion biofilms functioning simultaneousnitrification and denitrification. In Microbial ecology and water engineering & biofilms specialist groups(MEWE2016) (pp. 76-77). Copenhagen, Denmark : IWA.0/"!4!/!!26!.!4\6)M_6)DZXY^D02!/)(!/D!/.4,Session 7: Beneficial biofilms in Water Engineering   IWA Specialist Conference Microbial Ecology and Water Engineering 2016:  A joint conference of the MEWE and Biofilm specialist groups 76 Spatial distribution of microbial community and N2O depth profiles in counter- and co- diffusion biofilms functioning simultaneous nitrification and denitrification A. Terada*§, C. T. Kinh*, T. Suenaga*, T. Hori**, S. Riya*, B. F. Smets***, M. Hosomi* * Department of Chemical Engineering, Tokyo University of Agriculture & Technology, Naka, Koganei, Tokyo 184-8588, Japan (§Corresponding: akte@cc.tuat.ac.jp) ** Environmental Management Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Onogawa 16-1, Tsukuba, Ibaraki 305-8569, Japan *** Department of Environmental Engineering, Technical University of Denmark, Miljoevej, 2800 Lyngby, Denmark Keywords: Biofilm, High-throughput sequencing, Microbial community, Nitrous oxide (N2O) Introduction Redox stratification in a biofilm where both oxic and anoxic environments are present provides an opportunity for occurrence of simultaneous nitrification and denitrification (SND). Broader application of SND as a small footprint biological nitrogen removal system is contingent on surmounting technical challenges, e.g., control of oxygen supply and mitigation of nitrous oxide (N2O) emission. However, a conventional SND system faces a difficulty to control oxygen. Furthermore, N2O emission during SND is favored when oxygen supply for nitrification and organic carbon supply for denitrification are limited (Kampschreur et al. 2009). A conventional biofilm geometry (co-diffusion biofilm), where oxygen and electron donors, i.e., NH4+ and organic carbon, co-diffuse from the same direction, affords regions of oxygen-limited and organic carbon-limited conditions, thereby increasing N2O emission. A biofilm grown on a gas-permeable membrane, a.k.a., counter-diffusion biofilm, promises to overcome the challenges. This configuration allows precise oxygen supply from a gas-permeable membrane on which a biofilm grows (Syron and Casey 2008). NH4+ and organic carbon are supplied from the top of the biofilm whereas oxygen from the bottom, creating an environment where organic carbon and NO2-/NO3-, oxidized by ammonia oxidation at an aerobic region, are confronted. This counter-diffusion biofilm geometry for SND should be, therefore, of advantage to facilitate denitrification including N2O reduction.  To better understand a mechanism of N2O mitigation in the counter-diffusion biofilm for SND, this study compared depth profiles of N2O concentration and spatial distribution of microbial community in the biofilms employing counter- and co- diffusion geometries.  Material and Methods Two flow-cell reactors with an effective volume of 200 mL were operated for 3 months. One reactor (counter-diffusion biofilm) supplied air via one side of a flat-sheet silicone membrane to the other side in the presence of biofilm whereas the other reactor (co-diffusion biofilm) did from a bundle of hollow-fibers suspended in the bulk liquid. The co-diffusion biofilm had the same geometry with counter-diffusion biofilm including a silicon membrane for biofilm formation; however, an impermeable material was inserted under the membrane, ensuring no oxygen entry from the biofilm bottom. Synthetic medium consisting of CH3COONa and (NH4)2SO4 with concentrations of 200 mgC/L and 200 mgN/L was continuously fed to both reactors at an identical hydraulic retention time (14.5 h). The matured biofilms in both reactors were subjected to O2 and N2O microsensor measurements (n > 5) with the same methodology previously described (Terada et al. 2010). Subsequently, Session 7: Beneficial biofilms in Water Engineering   IWA Specialist Conference Microbial Ecology and Water Engineering 2016:  A joint conference of the MEWE and Biofilm specialist groups 77 they were excised and cryo-sectioned with 100 μm interval by a microtome. Each fraction of biofilm was subject to high-throughput Illumina sequencing of 16S rRNA gene amplicons (Aoyagi et al. 2015). Prediction of denitrifying functional genes from the sequencing libraries was performed by a bioinformatics tool PICRUSt (Langille et al. 2013). Results and Conclusions Nitrogen removal efficiencies of the counter- and co- diffusion biofilms were 73.5 ± 4.9% and 63.1 ± 4.0%, respectively, indicating better SND performance of the counter-diffusion geomertry (data not shown). The biofilm depth profiles of dissolved O2 and N2O concentrations (Figure 1) demonstrated that the bulk dissolved N2O concentration in the counter-diffusion biofilm (0.011 mgN/L) was 130 times lower than that in the co-diffusion biofilm (1.44 mgN/L). The far less N2O production in the counter-diffusion biofilm is congruent with the previous work (Pellicer-Nacher et al. 2010). High-throughput sequencing of 16S rRNA gene amplicons indicate that the classes Alphaproteobacteria, Betaproteobacteria, Actinobacteria and Crostridia were present in both biofilms. The family Nitrosomonadaceae was detected at the aerobic regions. The conversion of the phylogenetic information into functions by PICRUSt suggest that the genes encoding nitrite reductase (nirK), nitric oxide reductase (norB) and N2O reductase (nosZ) were higher in the middle of the counter-diffusion biofilm whereas higher at both edges of the co-diffusion biofilm (Figure 2). Taken together, biofilm geometry may lead to different spatial distribution and compositions of bacterial guilds responsible for N2O production and consumption, providing significant difference in N2O emission.     Figure 1 Depth profiles of dissolved O2 and N2O concentrations in (a) the counter- and (b) co- diffusion biofilms.       Figure 2 Gene counts of denitrification and relative gene abundance of N2O consumption over production as a function of biofilm depth with (a) the counter- and (b) co- diffusion geometries. References Aoyagi et al. (2015) Dynamic transition of chemolithotrophic sulfur-oxidizing bacteria in response to amendment with nitrate in deposited marine sediments. Front. Microbiol. 6, 426. Kampschreur et al. (2009) Nitrous oxide emission during wastewater treatment. Water Res. 43(17), 4093-4103. Langille et al. (2013) Predictive functional profiling of microbial communities using 16S rRNA marker gene sequences. Nat. Biotech. 31(9), 814-821. Pellicer-Nacher et al. (2010) Sequential aeration of membrane-aerated biofilm reactors for high-rate autotrophic nitrogen removal: Experimental demonstration. Environ. Sci. Technol. 44(19), 7628-7634. Syron and Casey (2008) Membrane-aerated biofilms for high rate biotreatment: Performance appraisal, engineering principles, scale-up, and development requirements. Environ. Sci. Technol. 42(6), 1833-1844. Terada et al. (2010) Inoculum effects on community composition and nitritation performance of autotrophic nitrifying biofilm reactors with counter-diffusion geometry. Environ. Microbiol. 12(10), 2858-2872. 0 500 1000 150000.010.020.030.0400.511.522.53N2O concentration [mgN/L]DO concentration [mg/L]N2ODOBiofilm depth [  m]0 500 1000 150000.511.522.5300.511.522.53N2O concentration [mgN/L]DO concentration [mg/L]N2ODOBiofilm depth [  m](a) (b)μ μ0.00.20.40.60.81.005000100001500020000250003000035000Gene count [-]Biofilm depth [µm]norB nirK nosZ nosZ/norBnosZ/norB[-]0.00.20.40.60.81.005000100001500020000250003000035000Gene count [-]Biofilm depth [µm]norB nirK nosZ nosZ/norBnosZ/norB[-](a) (b)

Spatial distribution of microbial community and N<sub>2</sub>O depth profiles in counter- and co- diffusion biofilms functioning simultaneous nitrification and denitrification

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Spatial distribution of microbial community and N<sub>2</sub>O depth profiles in counter- and co- diffusion biofilms functioning simultaneous nitrification and denitrification

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