2014 Abstract of Master Thesis Ecophysiology of bacterium ‘ japonica’ Keisuke MIZUTO Candidate for the Degree of Master Supervisor: Satoshi OKABE Division of Environmental Engineering

1. Introduction primer walking and Sangar method. Gene prediction Anaerobic oxidation (anammox) is analysis is conducted by following method (CDS microbially mediated process that produces gas prediction : MetaGeneAnnotator(ver1.0), tRNA + - (N2) using NH4 as electron donor and NO2 as electron prediction : tRNAScan-SE(ver1.23), rRNA prediction : acceptor [1]. The first anammox bacterial cultures were blastn(ver2.2.18)、RNAmmer(ver1.2)). enriched from wastewater treatment plant [2]. Therefore Confirmation of NO and N2O productions: To the initial focus of anammox research was on the confirm that ‘Ca. S. japonica’ use NO as anammox application of these . However, it soon became intermediate, and produce N2O from NO, activity test clear that anammox bacteria are responsible for a using crude extract of ‘Ca. S. japonica’ was performed. significant portion of nitrogen loss from oxygen The cell suspensions were treated with French press, and minimum zomes (OMZs) where up to half of global crude cell extract was obtained. Cell crude was marine nitrogen loss take place [3]. transferred to 5 ml serum bottles, resuspended in To date, at least six genera of anammox bacteria have phosphate buffer. The vials were made anoxic by 15 - been enriched and described. Among these, the deepest alternately applying under-pressure and He. NO2 (2.5 branching anammox genus, ‘Candidatus Scalindua’, is mM), and PTIO (1 mM) are added and incubated. During 31 46 the only representative found in all marine environments incubation, NO and N2O concentration in head space investigated worldwide. Therefore, they are considered gas was measured by GC/MS with time. Samples as marine anammox species. Furthermore, they have without crude extract and samples without crude extract high salinity tolerance (more than 30 ‰) and low growth and PMS were also prepared as controls. temperature (around 20 °C) [4]. Therefore, salinity and Systematic competition in MBR: Three MBRs were temperature are considered as important factors deciding established to observe systematic competition of their ecological niche. However physiological anammox bacteria in salinity condition. Three anammox information is still very limited due to the lack of bacteria, ‘Ca. S. japonica’, ‘Ca. Brocadia sinica’ and ‘Ca. enlichment anammox culture. Therefore, further Jettenia caeni’ were incubated in one reactor, and physiological study is important for a better population shift was observed with real time quantitative understanding of ecophysiology of ‘Ca. Scalindua’. PCR assay. Only difference among reactors is salinity In our laboratory, anammox bacterium ‘Candidatus concentration, reactor S1, S10 and S30 was salinity 1 Scalindua japonica’ was successfully enliched. Therefore, ppm, 10 ‰, and 30 ‰ respectively. Working volumes of the objective of this study is to investigate the the culture were maintained at 600 mL. A hollow-fiber physiological and ecological characteristics of ‘Ca. S. membrane unit was submerged into the culture and japonica’. To achieve this goal, we tried two approaches. operated at 25 oC. The hydraulic retention time was 1.9 First part is metagenomic and proteomic analysis. In this days and was decreased gradually to control nitrogen + - part we construct genome assembly of this bacterium, loading rate. Substrate (NH4 and NO2 ) concentration analyze protein expression comprehensively and was fixed at 1 mM respectively. Sludge retention time demonstrated estimated pathway. Second part is was not controlled in all three reactors. population dynamics study. In this pert, Systematic 3. Results and Discussion competition of three anammox genera in salinity Part 1. Metagenomic and Proteomic analysis for condition was observed, and Discussed the influence of ‘Candidatus Scalindua japonica’ salinity strength on ecological niche of ‘Ca. Scalindua’ Metagenome and proteome analysis: 47 contigs, 2. Materials and Methods 4,812,854 bp genome sequence are obtained by Biomass origin: ‘Ca. S. japonica’ is cultured in MBR metagenome analysis. Detail of genome data is shown in using 0.1 µm hollow fiber membrane, fed with water Table 1. Because total number of base of reads saturated containing sea salt and the substrates annomium, when number of base of reads exceed 100Mb, it is respectively 168 mg-N/L. MBR is operated in 20 ℃, considered that this genome sequence is covering whole HRT 5.1 days. Fluorescence in-situ hybridization ‘Ca. S. japonica’ genome. Completeness of this genome confirmed that ‘Ca. S. japonica’ made up 90 % of assembly based on essential genes were 97.6 %. accumulated biomass. Therefore, this genome seems to be well decoded. In S. Metagenome analysis: Genomic DNA was isolated japonica genome 1762 from biomass, was served to fragment and 8 kb pair end Table 1. overview of genome assembly CDSs were expressed. sequence in GS-FLX (Roche), and sequence reads were "Candidatus Scalindua japonica" In highly expressed obtained. This sequence reads were assembled by GS de Total size, bases 4.81Mb genes in ‘Ca. S. novo assembler ver 2.6, and 1 scaffold was obtained. Contigs 47 G+C contents （%） 38.8 japonica’ genome, From this scaffold, final genome sequence is decided by CDSs 4,019 genes involved in rRNA [16s-23s-5s] x1 tRNA 44 Completeness (%) 97.6

Fig 1. Production of NO and N2O anammox pathway and CO2 fixation were found. In this research, we could obtain com- prehensive knowledge about genes rerated to anammox metabolisms such as ATP synthesis, carbon fixation, respiratory chain and substrate transporter. Here, we focused on the genes related to anammox process. This is considered as a set Fig 2. Population dynamics of three anammox species in salinity of three reactions: (i) reduction of nitrite to nitric condition: A is salinity 1 ppm, B is salinity 10 ‰, C is salinity oxide by a cd1 nitrite reductase (NirS), (ii) condensation 30 ‰ of ammonium and into hydrazine by a anammox activity has gradually lost. Population change hydrazine synthase (HZS), and (iii) oxidation of was not observed in this reactor. It seems that hydrazine into N2 by a hydrazine dehydrogenase (HDH). mesohaline condition is not suitable for all anammox This pathway was proposed from the genome of ‘Ca. species. Kuenenia stuttgartiensis’ [5]. However resent research Nitrite concentration in the reactor S 30 gradually showed versatility of anammox pathway. The genome of increased. Finally, consumption of substrate was ‘Ca. Scalindua profunda’ also contained NirS [6]. completely stopped, First two weeks, growth of three However, the genome of ‘Ca. Jettenia caeni’ contained species was observed. From these results, we concluded copper-containing nitrite reductase (NirK) instead of that high salinity condition is most suitable for ‘Ca. S. NirS [7], the genome of ‘Ca. Brocadia sinica’ contained japonica’. no nitrite reductase [Oshiki et al, unpublished]. In our 4. Conclusion genome, the gene encodes for NirS was found. ‘Candidatus Scalindua japonica’ was successfully Furthermore, this genome contained several NorV genes. enriched. The genome assembly that had 4.8 Mb and 47 Therefore, we hypothesized that ‘Ca. S. japonica’ reduce contigs was obtained. Based on this genome, proteome nitrite using NirS and produce NO as an intermediate, analysis was performed. In this genome, nirS gene and and produce N2O from NO. several norV genes were found. Production of NO and Confirmation of NO and N2O productions: Time N2O by ‘Ca. Scalinua japonica’ was demonstrated by 31 46 course change of NO and N2O concentration is shown activity test with crude extract. Systematic competition 15 - in Fig 1. In the samples containing NO2 , crude extract of anammox bacteria in salinity condition concluded that 31 46 and PMS, production of NO and N2O was observed. ‘Ca. Scalinua japonica’ could not alive in almost low NO and N2O production activities were 0.45 and 0.55 condition, and high salinity condition was most suitable -1 -1 nmol-N mg-protein min respectively. On the other for ‘Ca. Scalinua japonica’. hand, in control samples, the samples without crude 15 - extract and the samples containing only NO2 , any Reference 31 46 NO and N2O gas was not observed. This means crude [1] Broda, E1. "Two kinds of missing in nature." Zeitschrift extract of ‘Ca. Scalindua japonica’ cell obviously für allgemeine Mikrobiologie 17.6 (1977): 491-493. contributed to NO and N O production. Therefore, we [2] Strous, Marc, et al. "Missing identified as new 2 planctomycete."Nature 400.6743 (1999): 446-449. concluded that ‘Ca. Scalindua japonica’ can produce NO [3] Jensen, Marlene M., et al. "Intensive nitrogen loss over the Omani and N2O. Shelf due to anammox coupled with dissimilatory nitrite reduction Part 2. Population dynamics induced by salinity to ammonium." The ISME journal 5.10 (2011): 1660-1670. [4] Awata, Takanori, et al. "Physiological characterization of an strength anaerobic ammonium-oxidizing bacterium belonging to the Systematic competition in MBR: Microbial population “Candidatus Scalindua” group."Applied and environmental dynamics of three anammox species was shown in Fig 2. microbiology 79.13 (2013): 4145-4148. Nitrite concentration in the reactor S1 reached [5] Strous, Marc, et al. "Deciphering the evolution and metabolism of an anammox bacterium from a community undetectable level soon after the start-up of the reactor. genome." Nature 440.7085 (2006): 790-794. Clear decreasing trend of ‘Ca. Scalindua japonica’ was [6] van de Vossenberg, Jack, et al. "The metagenome of the marine observed. Other two species maintain their concentration. anammox bacterium ‘Candidatus Scalindua profunda’illustrates It seems that low salinity condition was not suitable for the versatility of this globally important bacterium." Environmental microbiology 15.5 (2013): 1275-1289. ‘Ca. Scalindua japonica’ [7] Hira, Daisuke, et al. "Anammox organism KSU-1 expresses a Nitrite concentration in the reactor S10 kept NirK-type copper-containing nitrite reductase instead of a undetectable level first one week, but after that, NirS-type with cytochrome cd 1." FEBS letters 586.11 (2012): 1658-1663.