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Environmental Microbiology (2014) 16(10), 3030–3040

doi:10.1111/1462-2920.12248

Isolation of sublineage I Nitrospira by a novel cultivation strategy

Hirotsugu Fujitani,1 Norisuke Ushiki,1 Satoshi Tsuneda1* and Yoshiteru Aoi2,3** 1 Department of Life Science and Medical Bioscience, Waseda University, 2-2 Wakamatsu-cho, Shinjuku-ku, Tokyo 162-8480, Japan. 2 Institute for Sustainable Science and Development, Hiroshima University, 2-313 Kagamiyama, VBL403, Higashi-Hiroshima, Hiroshima 739-8527, Japan. 3 Department of Biology, Northeastern University, 360 Huntington Ave, Mugar Lifescience Bodg. 313, Boston, MA 02115, USA. Summary Nitrification is an important process in the biogeochemical nitrogen cycle and is widely exploited in biological wastewater treatment. Recently, Nitrospira has been recognized as the numerically dominant nitrite-oxidizing bacterial genus and is primarily responsible for the second step of aerobic nitrification. Nevertheless, the physiological properties of Nitrospira remain poorly understood because the organisms are difficult to isolate and culture. Here, we report a novel cultivation strategy for obtaining members of the Nitrospira sublineage I in pure culture. The method combines: (i) selective enrichment of Nitrospira using a continuous feeding reactor and (ii) purification followed by subcultivation via a cell sorting system by focusing on the unique characteristics of Nitrospira forming spherical micro-colonies. This strategy is potentially applicable to other uncultured or unisolated Nitrospira and could accelerate the physiological and biochemical understandings of this important group of organisms. Introduction Nitrification is a key process in the biogeochemical nitrogen cycle and is widely applied to biological wastewater Received 26 September, 2012; revised 5 August, 2013; accepted 9 August, 2013. For correspondence. *E-mail [email protected] waseda.jp; Tel. (+81) 3-5369 7325; Fax (+81) 3-3341 2684. **E-mail [email protected] or [email protected]; Tel. (+81) 82 424 7892; Fax (+81) 82 424 5732.

© 2013 John Wiley & Sons Ltd and Society for Applied Microbiology

treatment. Nitrite-oxidizing bacteria (NOB) catalyse the second step of nitrification – the oxidation of nitrite (NO2−) to nitrate (NO3−). Traditionally, the genus Nitrobacter (in the class alphaproteobacteria), which inhabits a range of environments, has been regarded as main representative of NOB. Indeed, Nitrobacter strains are more frequently isolated compared with other NOB (e.g. Bock et al., 1983; 1990; Sorokin et al., 1998). Therefore, the physiological and biochemical properties of NOB have been characterized by observations of Nitrobacter strains (Starkenburg et al., 2011). More recently, cultivation-independent molecular methods (Wagner et al., 1996; Burrell et al., 1998; Hovanec et al., 1998; Juretschko et al., 1998) and immunological techniques (Bartosch et al., 1999) have indicated that the genus Nitrospira is more widespread than Nitrobacter and most likely plays a primary role in the global nitrogen cycle, and natural nitrite conversion is an important part of the global nitrogen cycle. The genus Nitrospira represents a monophyletic lineage within the deep-branching bacterial phylum Nitrospirae, which is distinct from that of proteobacterial NOB (Ehrich et al., 1995). Despite their ecological and industrial importance, Nitrospira are usually recalcitrant to cultivation under laboratory conditions, and limited success has been achieved in isolating pure cultures (Watson et al., 1986; Ehrich et al., 1995; Lebedeva et al., 2008; 2011; Keuter et al., 2011). Although the ecology and eco-physiology of Nitrospira have been gradually clarified by molecularbased studies (Wagner et al., 1996; Burrell et al., 1998; Hovanec et al., 1998; Juretschko et al., 1998; Daims et al., 2001), the lack of a pure culture has limited our physiological understanding of the Nitrospira. In a recent successful attempt, a strain belonging to Nitrospira sublineage I was enriched from activated sludge and was partially characterized focusing on the essential physiological and cellular characteristics (Spieck et al., 2006). This strain was named ‘Candidatus Nitrospira defluvii’ and is regarded as a key NOB in sewage treatment processes. Moreover, an active chlorite dismutase in this microorganism was identified and validated by environmental genomics approach to sequence and assemble a 137 kbp-long genome fragment in combination with heterologous gene expression in Escherichia coli (Maixner et al., 2008) and structurally as well as

Isolation of uncultured Nitrospira biochemically characterized (Kostan et al., 2010). The following complete genomic analyses revealed the key metabolic pathways of Nitrospira, its differences to Nitrobacter and its evolution (Lücker et al., 2010). However, a representative pure strain of Nitrospira sublineage I remains to be identified. Here, we report the development of a novel cultivation strategy and its successful application to the cultivation of a previously unisolated sublineage I Nitrospira strain. The method combines two steps: first, selective enrichment of Nitrospira by a continuous feeding reactor was performed; and second, purification followed by sub-cultivation of the purified strain via a cell sorting system was performed. The method enabled culturing and characterization of the first isolate of Nitrospira sublineage I. Results Nitrospira enrichment In this study, Nitrospira enrichment cultivation was established and successfully applied as a step towards isolation (Fig. 1). A fixed bed continuous feeding bioreactor which has previously been designed was used in this study with similar running procedure (Fujitani et al., 2013). The activated sludge sampled from the sewage treatment plant was used as primary inocula. During the cultivation, nitrite concentration in the inlet medium solution or the flow rate, that is a volumetric feeding rate of nitrite, was gradually raised in response to increase in total nitrite oxidation rate per reactor volume (Fig. 2A). Maximum concentration of nitrite in the inlet medium was set 5.7 mM, whereas the nitrite concentration was kept lower than 0.14 mM with a few times of exception. In this manner, nitrite concentration in the reactor could be controlled and maintained at the desired low level while providing higher concentration of nitrite as inlet medium. The ratio of Nitrospira to the total microorganism population was monitored by fluorescence in situ hybridization (FISH) direct counting using Nitrospira-specific probes throughout the experiment. The ratio, which was less than 1% in the inoculum, gradually increased as the feeding rate of nitrite increased (Fig. 2B). Conversely, the nitrite concentration in the reactor was kept constant (below 0.14 mM) during the enrichment process (Fig. 2A). After 342 days, both the Nitrospira ratio and the total dry biomass had attained high levels (69.3% to the total bacterial cells and 0.4–0.8 g l−1 as total dried biomass amount). Thereafter, a portion of enrichment cultures were transferred to a secondary bioreactor for replicate samples, in which the high abundance of Nitrospira persisted over 2 years (approximately 80% on average, and 96% at the maximum by FISH direct counting). In contrast, Nitrobacter, considered as a critical competitor of

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Nitrospira (Nogueira and Melo, 2006; Blackburne et al., 2007; Graham et al., 2007), was not detected by FISH throughout the experiment. In the enrichment culture, Nitrospira formed various sizes of spherical micro-colonies (clumps of small densely packed cells), as has been often reported in previous studies (Juretschko et al., 1998; Bartosch et al., 1999; Daims et al., 2001; Spieck et al., 2006). Although the average size of micro-colonies varied considerably in this study, the distribution of micro-colony diameter was relatively consistent at 5–20 μm. High-throughput separation of Nitrospira by cell sorting Here, a new cell-sorting method for separating target microorganisms was established and successfully applied to the Nitrospira enrichment culture (Fig. 1). Although Nitrospira could be enriched until its proportion in the microbial community exceeded 80%, other types (mostly heterotrophs) were strongly resistant to elimination by the cultivation-based enrichment process. Separation of the Nitrospira population was a challenging issue following the enrichment step. As described above, Nitrospira cells tended to form densely packed spherical clumps in the enrichment culture and became embedded in relatively large microbial flocs or biofilm, where they co-inhabited with other microbes. These flocs or biofilm was readily dispersed by weak sonication, yielding relatively small particles. Following sonication, a range of particle sizes was observed in the samples. These were classified into three types by microscopic observation: (i) dense spherical microcolonies (diameter: 3–20 μm); (ii) uneven-shaped microbial clumps (diameter: > 3 μm) and (iii) planktonic single cells (diameter < 1 μm). FISH using Nitrospiraspecific probes indicated that most dense spherical micro-colonies contained pure Nitrospira cells, whereas uneven-shaped clumps contained multiple types of microorganisms. Small numbers of Nitrospira cells were found within the planktonic group. On the basis of these differences in particle size and shape, we conceived that Nitrospira could be separated from other cell types by using a cell sorting system (Fig. 1). A common use of the cell sorting system is to physically sort micro-particles based on their lightscattering or fluorescent properties in order to purify populations of interest. In this experiment, forward scatter (FSC) and side scatter (SSC) were used for particle sorting. In principle, the magnitude of FSC and SSC signals correlates with the size of the particles and their complexity respectively. Within the enrichment sample, (i) dense spherical micro-colonies (considered as Nitrospira) exhibited relatively high FSC and low SSC signals; (ii) uneven-shaped microbial clumps (containing multiple

© 2013 John Wiley & Sons Ltd and Society for Applied Microbiology, Environmental Microbiology, 16, 3030–3040

3032 H. Fujitani, N. Ushiki, S. Tsuneda and Y. Aoi

Fig. 1. Advanced cultivation and purification strategy for the isolation of Nitrospira. The Nitrospira from activated sludge are selectively enriched within a continuous feeding bioreactor (phase I). Nitrospira micro-colonies in enrichment samples are separated by cell sorting and inoculated into 96-well microtiter plates containing nitrite medium (phase II). Growth-positive strains identified as Nitrospira are transferred and upscaled for investigation of their physiological characteristics (phase III).

© 2013 John Wiley & Sons Ltd and Society for Applied Microbiology, Environmental Microbiology, 16, 3030–3040

Isolation of uncultured Nitrospira

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Fig. 3. Separation of Nitrospira micro-colonies samples via cell sorting. Flow cytometric analysis of Nitrospira enrichment samples was conducted. Identified dot plot area was divided into four sub-areas (P1–P4). Fractions separated from each sub-area were subjected to microscopic observation.

Incubation time [days]

Fig. 2. Enrichment culture of Nitrospira by a continuous feeding bioreactor. A. The dotted line represents a volumetric feeding rate of nitrite; the open square represents nitrite–nitrogen concentration in the bioreactor effluent. B. The ratio of Nitrospira to total microorganisms obtained by FISH analysis with direct counting.

microorganisms) exhibited relatively high levels of both signals because of their large size and rough surface, and (iii) planktonic cells exhibited relatively low levels of both signals. Applying the concept described above, Nitrospira micro-colonies were distinguished and physically separated from the particle mixture by their distinctive lightscattering signature through the cell sorting system without specific labelling. Initially, the enrichment sample taken from the secondary bioreactor on day 413 (following transfer of enrichment cultures) was sonicated and applied to the cell sorter. On this date, the ratio of Nitrospira to the total

microorganisms was 84% by FISH direct counting (Fig. S1). The samples were applied at a flow rate of 200–300 events s−1 operating in single cell mode, and the dot plot area was identified (Fig. 3). The dot plot displays fractions sorted out from total populations and observed by FISH microscopy. As expected from the above hypothesis, the P4 area in the dot plot contained most of the pure Nitrospira micro-colonies (Fig. 4C). Planktonic cells and small micro-colonies tended to occupy the P1 and P2 areas (low FSC fraction). The P3 area included larger and more complex multi-species aggregates (Fig. 4B). In order to indicate quantitatively the advantage of this sorting method, the ratios of Nitrospira cells to initial growth unit before and after sorting were calculated with FISH analysis and direct counting methods. The ratio of Nitrospira cells to the total number of microbial cells in enrichment samples prior to sorting was 84% (Figs 4A and S1). However, the ratio of Nitrospira micro-colonies to the number of total initial growth units always remained

Fig. 4. Nitrospira micro-colonies collected from fractions in the dot plot area. The yellow cells are Ntspa662-stained Nitrospira and the green cells are SYTOX green-stained other microorganisms. A. Enrichment samples treated by sonication prior to extraction of Nitrospira micro-colonies. B. Multi-species colonies obtained from P3. C. Nitrospira micro-colonies obtained from P4. All scale bars are 5 μm.

© 2013 John Wiley & Sons Ltd and Society for Applied Microbiology, Environmental Microbiology, 16, 3030–3040

3034 H. Fujitani, N. Ushiki, S. Tsuneda and Y. Aoi below 1% (Figs 4A and S1). This is because, even after dispersion of the sample, most of Nitrospira clumps remained as a form of micro-colonies consisting of several dozen to hundreds of cells, whereas comparatively a large number of free single cells (mostly other types) were produced. After sorting and separation, this ratio increased to 32% or 99% of individual cells (Fig. 4C and Fig. S1). Pure culture and characterization Single micro-colonies (Nitrospira micro-colonies) collected by cell sorting of particles from the P4 area were individually inoculated into 96-well microtiter plates containing a low nitrite-concentration medium (0.14– 0.71 mM). In total, 145 wells were inoculated. After 1 month of incubation under dark and static conditions, Nitrospira growth was confirmed in five wells by microscopic observation. FISH analysis and 16S ribosomal RNA (rRNA) sequences analysis revealed that each culture was related to Nitrospira. The growth of other types, the growth of multiple species or no growth was found in other wells. These results were shown in supplementary data (Fig. S2 and Table S1). Nitrospira pure cultures grown in the wells were transferred into 5 ml vials containing 1 ml fresh nitrite medium, after which growth was upscaled to 100 ml plastic tubes containing 20 ml fresh nitrite medium, and finally, to 2 l Erlenmeyer flasks containing 1 l fresh nitrite medium (Fig. 1).

The near full-length 16S rRNA gene sequence shared a high level of identity with the sequences of Ca. N. defluvii (DQ059545) (99.8%) (Spieck et al., 2006), uncultured Nitrospira sp. clone 2 (DQ414438) (100%) and clone OWP detected from the enrichment samples used in the previous study (Fujitani et al., 2013) (100%) (Fig. 5). Sequence similarities of the five strains with each other were 99.8–100%. Strain ND1 isolated in this study, which originates from the activated sludge of a wastewater treatment and is one of five pure cultures, was identified as a suitable sublineage I. The purity of the culture was carefully inspected throughout the experiment by several methods: (i) FISH microscopic observation, (ii) transferring of cultures to several types of organic culture medium and (iii) polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) analysis. No contaminating cells were observed in these cultures with FISH. Especially the inner part of micro-colonies assembled in pure culture was carefully inspected by confocal laser scanning microscopy to further validate the purity. Each micro-colony was found to solely comprise uncontaminated Nitrospira cells (Fig. 6C). Purity of the cultures was also repeatedly checked in successive transfers to both agar-plate and liquid media containing 200-fold diluted Nutrient Broth (DNB) (BD, Franklin Lakes, NJ, USA) and R2A (DR2A). No growth was observed in or on any types of culture medium. PCR-DGGE analyses followed by DNA extraction and sequencing were applied to check contamination (Fig. S3). As a result, only one single band Fig. 5. Phylogenetic analysis showing the affiliation of the Nitrospira isolate in this study. The phylogenetic tree is based on 16S rRNA gene sequences of selected Nitrospira. The tree was constructed using the neighbour-joining algorithm. Numbers at the branch nodes are bootstrap values. Previously defined (Daims et al., 2001) sublineages of the genus Nitrospira (Roman numbers) are shown. The scale bar corresponds to 10% estimated sequence divergence. The clones (OWP) stated in the phylogenetic tree were obtained in our previous study (Fujitani et al., 2013).

© 2013 John Wiley & Sons Ltd and Society for Applied Microbiology, Environmental Microbiology, 16, 3030–3040

Isolation of uncultured Nitrospira

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was obtained from pure strain ND1 and was affiliated to sublineage I Nitrospira (Fig. S3 line 2). An identical band was also observed from the sample obtained from the enrichment culture used as a source (Fig. S3 line 1). Strain ND1 demonstrated clear nitrite oxidation activity in liquid batch culture (Fig. S4). The strain consumed approximately 1.43 mM nitrite within 11 days at 23°C, with equivalent production of nitrite. By incubating the strain under conditions of varying nitrite concentrations, the optimal nitrite concentration for growth of strain ND1 was shown to lie between 0.71 and 2.14 mM (Fig. S5). This range corresponded approximately to nitrite concentration requiring enrichment of Ca. N. defluvii (Spieck et al., 2006). The optimal temperature for strain ND1 was investigated by incubation in medium containing 1.43 mM of nitrite across the temperature range 10–46°C. Nitrite-oxidizing activity was observed between 10 and 34°C inclusive, with a wide optimal temperature range of 25–31°C (Fig. S6). Under scanning electron microscopy (SEM) and fluorescence microscopy, the pure cultures of Nitrospira were observed to form dense micro-colonies as observed in the enriched sample (Fig. 6A and C). Micro-colonies grew botryoidally with uniform shape and with diameters ranging from approximately 3 to 20 μm. Transmission electron microscopy (TEM) observations of ultrathin sections of a microcolony revealed that individual cells are irregularly shaped, resembling those of Nitrospira moscoviensis and Ca. N. defluvii, which form star-like extensions of their outer membrane (Fig. 6B) (Ehrich et al., 1995; Spieck et al., 2006). The width and length of the spiraland rod-shaped cells ranged from 0.3 to 0.8 μm and from 0.6 to 1.0 μm respectively.

Discussion Enrichment Several factors must be considered in Nitrospira enrichment; these are (i) sensitivity to nitrite, (ii) competition with Nitrobacter and (iii) coexistence of heterotrophs.

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Fig. 6. Morphology of strain ND1 isolated from activated sludge. A. Scanning electron microscopic image of a micro-colony. Scale bar is 5 μm. B. Ultrathin section of a micro-colony revealing the wide periplasmic space and extracellular polymeric substances. Scale bar is 1 μm. C. Growing Nitrospira micro-colonies observed under confocal laser scanning microscopy. Micro-colonies cells are stained as described in FISH protocols. Scale bar is 10 μm.

Therefore, to acquire highly enriched culture of Nitrospira, a means of maximally enhancing the growth activity of Nitrospira while inhibiting its competitors are sought. Although previous studies have succeeded in repressing the growth of Nitrobacter cells and heterotrophic contaminants by the combination of using the medium containing low concentration of nitrite and addition of antibiotics (ampicillin), resulting in the selective enrichment of Nitrospira (Spieck et al., 2006), here, we propose an alternative approach. It is well known that cells of Nitrospira are sensitive to their substrate nitrite. Therefore, the growth activity of Nitrospira is inhibited at high nitrite concentrations over 5.71 mM (Wagner et al., 1996; Nogueira and Melo, 2006). Competition with other nitrite oxidizers such as Nitrobacter and Nitrotoga (Alawi et al., 2009) is another important issue. In this study, out-competing of Nitrobacter is essential for selective enrichment of Nitrospira. Early studies have demonstrated that the affinity for nitrite is higher in Nitrospira than in Nitrobacter; thus, Nitrospira are considered as k-strategists, whereas Nitrobacter are r-strategists (assuming that nitrite concentration is the major driver of the competition; Schramm et al., 1999; Wagner et al., 2002). Indeed, in this experiment, feeding low levels of nitrite into the reactor throughout the enrichment process proved effective at outcompeting Nitrobacter. It is generally known that a considerable number of various types of heterotrophic bacteria can coexist with nitrifying bacteria despite providing inorganic substrate which does not contain any organic matter. Nitrifying bacteria release organic metabolic by-products and cell lysate from dead cells, which provide carbon and energy sources for heterotrophs (Rittmann et al., 1994; Kindaichi et al., 2004; Okabe et al., 2005; Matsumoto et al., 2010). Therefore, minimizing heterotrophic growth is an important issue for the enrichment of Nitrospira. We hypothesized that the high flow rate condition suppresses heterotrophic growth by enhancing the outflow of soluble organic compounds produced by nitrite oxidizers in the reactor.

© 2013 John Wiley & Sons Ltd and Society for Applied Microbiology, Environmental Microbiology, 16, 3030–3040

3036 H. Fujitani, N. Ushiki, S. Tsuneda and Y. Aoi Periodic discharge of overgrown biomass might also effectively enrich Nitrospira by reducing the quantity of inactive cells, debris and extracellular polymeric substances in the biomass which constitute potential nutrient sources for heterotrophs. This concept was proven by the fact that Nitrospira were enriched efficiently in the continuous feeding bioreactor (constituting 96% of the total bacterial population at maximum) and that populations were stably retained (exceeding 80% of bacterial counts) for over 2 years. Cell sorter-based separation of micro-colonies To physically separate or to concentrate specific target cells from the heterogeneous mixtures such as environmental samples is an important issue for the recent microbiological research because it is effective for obtaining pure culture or analysing genomic or biochemical information of uncultivated microorganisms. Recently, cell sorting systems have widely been applied in the field of microbiology, leading successes to the phylogenetic, genomic and physiological studies. Among them, gel microdroplet (GMD) technologies combined with flow cytometric sorting have been reported as a high throughput cultivation method (Manome et al., 2001; Zengler et al., 2002). It has been proved that microbial growth inside GMDs as a form of micro-colony can be distinguished based on FSC and SSC signals (Zengler et al., 2002). Nitrospira usually form spherical single micro-colonies in environmental samples as well as other nitrifying microorganisms. By focusing on this unique characteristic, distinguishing and high throughput separation of Nitrospira micro-colonies from the other types of microbial types have been demonstrated based on the forward and side scattering signals. To the best of our knowledge, this is the first application of the cell sorting system to separate specific environmental microorganisms based on the micro-colony formation and to lead isolation of uncultured or unisolated microorganisms. In this study, Nitrospira were separated from mixed cultures containing untargeted populations and selectively inoculated into 96-well microtiter plates, exploiting the high throughput capacity and separation efficiency of the cell sorter. Although the proportion of Nitrospira cells in the enrichment sample became as high as 80% based on cell counting, the ratio of Nitrospira inoculated as initial growth units (regarding individual free-living single cell and micro-colony as an equivalent unit) is expected to be much lower (Fig. S1). This is because even after dispersion of the sample, most of Nitrospira clumps remained as a form of micro-colonies consisted of several dozen to hundreds of cells, whereas a comparatively large number of free single cells (mostly other

types) were produced. Indeed, the ratio of Nitrospira micro-colonies to the number of total initial growth units always remained below 1% (Figs 4A and S1). After sorting and separation, this ratio increased to 32.5% (or 99.9% of individual cells) (Figs 4C and S1). As the ratio of Nitrospira micro-colony (based on growth unit counting) in the enrichment sample cannot be increased higher than 1%, and it is difficult to remove coexisting heterotrophic microorganisms by other methods, this newly designed method has a strong advantage compared with the conventional method on efficiency of isolation. The total number of wells into which Nitrospira microcolonies had been inoculated was estimated as 145 out of 672 (22%). Other wells were expected to remain empty (no cells deposited) or inoculated with other types of microbes. However, Nitrospira growth was confirmed in only five wells following 1 month of incubation. In other words, a mere 3% (5 out of 145) of inocula had recovered during the incubation period. The growth was judged by direct microscopic observation using the sample which had been partially taken from each well. This required certain level of cell density (at least 105–106 per 1 ml) for the detection. Previous studies reported doubling time of Nitrospira as 0.5–4 days (Watson et al., 1986; Ehrich et al., 1995; Maixner et al., 2006). Thus, it is likely that the cell number in most wells was under the detection limit after the 30 days of incubation period. Being damaged by sonication and laser of cell sorter, and inhibition by eluted toxic material from plastic microtiter plates can be an another possible reason. The low recovery ratio also represents the difficulty of some types of Nitrospira to purify. Given this low recovery, it is apparent that increasing the ratio of target microbial type (Nitrospira in this study) and inoculating micro-colonies individually by our method would significantly increase the probability of success because the majority of Nitrospira inocula failed to recover despite being inoculated as single micro-colonies. Inoculation of Nitrospira as a form of a micro-colony has another advantage. Ensuring that Nitrospira were inoculated as micro-colonies rather than as single cells would also enhance the probability of growth in a particular well because the number of inoculated cells was dozens of times higher. Moreover, the dense packing of tens to hundreds of cells constitutes a ‘high local cell density condition’, which may be favourable for Nitrospira growth. Indeed, it has been reported that the growth or expression of specific activity of some types of microorganisms requires a certain cell density, probably because such activity is controlled by cell-to-cell chemical communication in a process known as quorum sensing (Mukamolova et al., 2006; Shah et al., 2008).

© 2013 John Wiley & Sons Ltd and Society for Applied Microbiology, Environmental Microbiology, 16, 3030–3040

Isolation of uncultured Nitrospira Characteristics of isolates

Experimental procedures

A series of microscopic observations confirmed the morphological characteristics of strain ND1 (Fig. 6). As frequently reported in early studies, Nitrospira tend to form clumps of small densely packed cells, which manifest as spherical micro-colonies of various sizes, in activated sludge and biofilm samples (Juretschko et al., 1998; Bartosch et al., 1999; Daims et al., 2001; Spieck et al., 2006). Interestingly, this tendency to form micro-colonies is retained under pure culture conditions, indicating that micro-colony formation is an intrinsic feature of strain ND1. However, during starvation, all cells in the pure culture reverted to a planktonic state (Spieck et al., 2006). This switching behaviour (aggregate vs. planktonic) of Nitrospira likely confers an adaptive advantage during occasional exposure to nitrite concentration changes, which ensures the survival of the organism. Temperature exerts a major influence on nitriteoxidizing activity and on the population structure of NOB in natural habitats and wastewater treatment plants (Alawi et al., 2009). The nitrite-oxidizing activity of strain ND1 exhibited a unique step-like temperature profile. The strain consumed nitrite across a broad temperature range (10–34°C; Fig. S6). The optimal temperature was broadly distributed from 25–31°C, and similar to that of Ca. N. defluvii enrichment culture 32°C as optimal temperature. (Spieck and Lipski, 2011). The representative pure sublineage II strain, Nitrospira moscoviensis, favours a much higher temperature for nitrite oxidation (39°C; Ehrich et al., 1995).

Continuous feeding bioreactor

Conclusive remarks A novel cultivation method was developed and successfully applied to the isolation of previously unisolated Nitrospira. First, the selective enrichment of Nitrospira was successfully demonstrated using a continuous feeding bioreactor. Second, Nitrospira were separated by cell sorting on the basis of the morphological characteristics of their micro-colonies. Single micro-colonies were inoculated individually into microtiter plate wells. Following long-term incubation in low-concentration medium and successive upscaling of the culture, the first isolate classifiable as Nitrospira sublineage I was successfully obtained. Interestingly, strain ND1 continued to form micro-colonies under pure culture conditions. The newly developed method could potentially be applied to generic cultivation of environmentally important uncultured microorganisms, which can be separated based on the properties of their micro-colonies. Such organisms include other nitrifying microorganisms (ammonia-oxidizing, nitriteoxidizing and anaerobic ammonia-oxidizing bacteria).

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The primary inoculum for the enrichment of Nitrospira was sampled from the nitrification stage of the municipal sewerage disposal plant at 15–16°C in Ochiai, Tokyo, Japan in February 2009. A continuous feeding bioreactor with biomass carrier comprising polyester non-woven fabric materials was set up to retain the bacteria in an active state (Fig. 1) as described in previous study (Fujitani et al., 2013). Dissolved oxygen concentration was around 6.0 mg l−1. The control of temperature and pH and the preparation of inorganic medium were conducted according to the protocol described previously (Fujitani et al., 2013). The influent nitrite–nitrogen concentration was periodically increased to 0.36 mM (days 0–200), 1.43 mM (days 201–276) and 5.71 mM (days 277–360). During the same time periods, hydraulic retention time was gradually shortened from 4.8 h to 1.6 h to enrich the Nitrospira. A portion of enrichment culture was transferred to a secondary bioreactor to provide sufficient culture for replicate sampling. The secondary bioreactor was stably operated for over 2 years.

Chemical Analyses Nitrite–nitrogen and nitrate–nitrogen concentrations were determined quantitatively by Ion Chromatography (IC 2001, Tosoh, Tokyo, Japan). Influent/effluent samples in the bioreactor and nitrite oxidation activity test samples were filtered through 0.20 μm cellulose acetate membrane filters (Advantec, Tokyo, Japan).

FISH and DNA staining All in situ hybridizations were performed following the standard protocol (Amann et al., 1990) in hybridization buffer at 46°C for 2.5 h. The applied oligonucleotide probes were listed in Supporting Information (Table S2). Oligonucleotides were synthesized and fluorescently labelled with a hydrophilic sulfoindocyanine dye (Cy3) or fluorescein isothiocyanate at the 5’ end (Tsukuba Oligo Service, Tsukuba, Japan). Separately from FISH with the EUB mix, SYTOX Green nucleic acid stain (Life Technologies, Carlsbad, CA, USA) was applied as a universal cellular stain. Stained cells were detected and recorded by a confocal laser scanning microscope (IX71, Olympus, Tokyo, Japan) and by a fluorescence microscope (Axioskop 2 plus, Carl Zeiss, Oberkochen, Germany).

Selective sorting of Nitrospira micro-colonies Nitrospira micro-colonies were successfully separated from untargeted microorganisms (multi-species micro-colonies, planktonic microbial cells) by a cell sorting system (FACS Aria, BD, Franklin Lakes, NJ, USA). The samples were extracted from the secondary bioreactor on Day 413 following transfer of enrichment cultures, dispersed by ultrasonic treatment (Sonifier II model 150, Branson, Danbury, CT, USA) for 1 min at intensity of dial 2 and filtered successively

© 2013 John Wiley & Sons Ltd and Society for Applied Microbiology, Environmental Microbiology, 16, 3030–3040

3038 H. Fujitani, N. Ushiki, S. Tsuneda and Y. Aoi through some filter papers (pore size 35 μm, BD) to remove large cell aggregates. Filtered samples were applied to a cell sorter, with sample flow rate adjusted to approximately 200–300 events s−1 in single cell mode for purity enhancement. The dot plot area defined on a two-parameter histogram (FSC vs. SSC) was identified. At least 10 000 particles were analysed for each histogram. Fractions separated from each region (P1–P4) of the dot plot area were mounted onto glass slides (at least 500 particles per slide) and were investigated by FISH analysis. In each fraction, the ratio of Nitrospira to total microbial cells was calculated by direct counting. Subsequently, a fraction containing mostly Nitrospira micro-colonies was identified. Samples within this fraction were sorted and inoculated into 96-well microtiter plates for sub-cultivation.

Cultivation of ND1 isolates Single micro-colonies of Nitrospira separated by a cell sorter were incubated for 1 month in 96-well microtiter plates containing a low nitrite-concentration medium (0.14– 0.71 mM). Medium composition other than NaNO2 corresponded to enrichment culture in the bioreactor, and the final pH was adjusted to between 7.5 and 7.8. Cultivation was conducted under dark and static conditions. Cell growth was assessed by FISH analysis and fluorescence microscopy. Pure bacterial strains grown in the 96well microtiter plates were identified by 16S rRNA gene sequences analysis. Novel strains unambiguously identified as Nitrospira were transferred into 5 ml vial tubes containing 1 ml of medium and were incubated as described above. Subsequently, they were transferred into 100 ml plastic tubes containing 20 ml of medium and later into 2 l Erlenmeyer flask containing 1 l of medium in order to investigate their physiological characteristics. During cultivation, nitrite consumption was frequently monitored by use of a water test kit (Kyoritsu Chemical Check Lab, Tokyo, Japan) and an ion chromatography. Cell growth was checked by microscopic observation. Nitrite-depleted cultures were supplied with fresh nitrite medium.

PCR amplification and seaquence of the 16S rRNA gene and phylogenetic analysis The 16S rRNA gene fragment (ca. 1500 bp) of the strain was amplified using the primers 27f/1492r. Amplification of the 16S rRNA gene was conducted according to the protocol described previously (Fujitani et al., 2013). Finally, the 16S rRNA gene sequences, comprising approximately 1500 bases, were determined. Alignment editing and phylogenetic analyses were performed using CHROMASPRO version 1.4.1 software (Technelysium Pty, Tewantin, Australia) and MEGA4.0 software (Tamura et al., 2007). The bacterial 16S rRNA sequences were compared with those available from the DNA Data Bank of Japan database (National Institute of Genetics, Mishima, Japan). All clones containing 16S rRNA genes with > 98% similarity to the above-characterized sequence were grouped into an operational taxonomic unit. Genetic distance was calculated using a p-distance model of nucleic acid substitution.

PCR-DGGE analyses DGGE analyses were performed according to the standard protocol (Muyzer et al., 1993). The partial 16S rRNA genes in pure culture were amplified by PCR with the primers 27f/ 1492r. The PCR products were purified and were diluted 1/100 with Tris-EDTA (TE) buffer and used as a template for second PCR with 907r and GC-clumped 341f primers. The second PCR was carried out using the following programme: 2 min at 95°C; 30 cycles of 1 min at 94°C, 1 min at 55°C and 1 min at 72°C; and 5 min at 72°C. DGGE was performed at 60°C and 130 V for 16 h with a gradient denaturants (30–70%).

Purity test The growth of heterotrophic contaminants was tested using both agar-plate and liquid media containing 200-fold DNB (peptone 1 mg l−1, meat extract 0.6 mg l−1) and DR2A (polypeptone 2.5 mg l−1, casamino acid 2.5 mg l−1, sodium pyruvate 1.5 mg l−1, soluble starch 2.5 mg l−1, yeast extract 2.5 mg l−1, KH2PO4 2.5 mg l−1, MgSO4 · 7H2O 0.25 mg l−1). The temperature was maintained at 23°C, and the cultivation was conducted under dark and static conditions for 3 weeks.

Nitrite oxidation activity test and optimal temperature screening Nitrite oxidation activity tests were performed at an incubation temperature of 23°C in 300 ml Erlenmeyer flasks filled with 100 ml mineral nitrite medium (1.43 mM). Media were stirred to ensure sufficient oxygen supply. Optimal nitrite concentration was performed in 50 ml test tubes filled with mineral nitrite medium (0.71–5.0 mM) during incubation of 3 days. Optimal temperature for nitrite concentration was investigated within the range 10–46°C in test tubes filled with 10 ml mineral nitrite medium (1.43 mM).

Electron microscopy Ultrathin sections of isolate were prepared and observed under TEM. Isolated cells were fixed in 2% glutaraldehyde in 0.1 M phosphate buffer, were collected and attached on 0.2 μm filters, and were post-fixed in 2% osmium tetroxide for 2 h at 4°C. The specimens were dehydrated in a graded series of ethanol, placed in propylene oxide and embedded in epoxy resin (EPON812) for 48 h at 60°C. Ultrathin sections were cut and stained with uranyl acetate and lead citrate prior to examination under TEM (JEM-1200EX, JEOL, Tokyo, Japan) at 80 kV. Images of isolate were obtained under SEM. Following fixation and dehydration, specimens were placed in isoamyl acetate, critical point dried, coated using an osmium plasma coater and examined under SEM (JSM-6320F, JEOL) at 5 kV.

Acknowledgements This research was supported by the Industrial Technology Research Grant Program (05A07508a) from the New Energy and Industrial Technology Development Organization of

© 2013 John Wiley & Sons Ltd and Society for Applied Microbiology, Environmental Microbiology, 16, 3030–3040

Isolation of uncultured Nitrospira Japan, the Sasagawa Scientific Research Grant from The Japan Science Society, and the Special Research Projects (2011B-200 and 2012B-171) from Waseda University.

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Supporting information Additional Supporting Information may be found in the online version of this article at the publisher’s web-site: Fig. S1. The ratios of Nitrospira to the total microbial types in two samples were compared: (i) enriched culture sample treated by weak sonication as shown as ‘original sample’ and (ii) the sample collected from cell sorter after application of the original sample in order to separate micro-colony from other populations, as shown as ‘sorted sample’. The ratios were estimated by FISH direct counting method. Two types of counting methods were performed: (i) simply counting all the cells and calculating the ratio as ‘the ratio based on microbial cell counting’ and (ii) counting free living cells and microcolonies individually as shown as ‘the ratio based on initial growth unit counting’. Both ‘a single cell’ and ‘a single microcolony’ were regarded as the equivalent unit and so were called ‘initial growth unit’. Fig. S2. Representative FISH images showing the growth of (A) Nitrospira, (B) Nitrospira with contaminants and (C) other microbial type in each well of 96 micro-titer plate after the 1 month of incubation. Ntspa662- and SYTOX green-doublystained cells (Nitrospira) are colored in yellow, whereas other types are coloured in green stained only by SYTOX green. All scale bars are 10 μm. Fig. S3. PCR-DGGE band profile of enrichment sample (lane 1) and pure strain ND1 (lane 2). Fig. S4. Nitrite consumption by strain ND1 grown at 23°C in Erlenmeyer flasks, plotted against nitrate production. The filled circle and filled square represent nitrite and nitrate concentrations respectively. Error bars indicate the standard deviation of the mean of triplicate measurements. Fig. S5. Optimal nitrite concentration for nitrite consumption by strain ND1. The inoculum was extracted from a pre-culture grown at 25°C. Optimal nitrite concentration was determined as that yielding the highest nitrite oxidation rate during 3 days of incubation. Error bars indicate the standard deviation of the mean of triplicate measurements. Fig. S6. Optimal temperature for nitrite consumption by strain ND1. The inoculum was extracted from a pre-culture grown at 25°C. Initial nitrite concentration was 20 mg-N l−1. Optimal temperature was determined as that yielding the highest nitrite oxidation rate during 3 days of incubation. Error bars indicate the standard deviation of the mean of triplicate measurements. Table S1. Sequence analysis of five pure cultures obtained in this study. Table S2. FISH probes used in this study.

© 2013 John Wiley & Sons Ltd and Society for Applied Microbiology, Environmental Microbiology, 16, 3030–3040

Isolation of sublineage I Nitrospira by a novel cultivation strategy.

Nitrification is an important process in the biogeochemical nitrogen cycle and is widely exploited in biological wastewater treatment. Recently, Nitro...
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