This present study aimed to characterize the bacterial community in a well-established nitrifying reactor by high-throughput sequencing of 16S rRNA amplicons. The laboratory-scale continuous stirred tank reactor has been supplied with ammonium (NH4+) as sole energy source for over 5 years, while no organic carbon has been added, assembling thus a unique planktonic community with a mean NH4+ removal rate of 86 ± 1.4 mg NH4+-N/(L d). Results showed a nitrifying community composed of bacteria belonging to Nitrosomonas (relative abundance 11.0%) as the sole ammonia oxidizers (AOB) and Nitrobacter (9.3%) as the sole nitrite oxidizers (NOB). The Alphaproteobacteria (42.3% including Nitrobacter) were the most abundant class within the Proteobacteria (62.8%) followed by the Gammaproteobacteria (9.4%). However, the Betaproteobacteria (excluding AOB) contributed only 0.08%, confirming that Alpha- and Gammaproteobacteria thrived in low-organic-load environments while heterotrophic Betaproteobacteria are not well adapted to these conditions. Bacteroidetes, known to metabolize extracellular polymeric substances produced by nitrifying bacteria and secondary metabolites of the decayed biomass, was the second most abundant phylum (30.8%). It was found that Nitrosomonas and Nitrobacter sustained a broad population of heterotrophs in the reactor dominated by Alpha- and Gammaproteobacteria and Bacteroidetes, in a 1:4 ratio of total nitrifiers to all heterotrophs.
Nitrification is an important process in wastewater treatments as it removes ammonium, a nitrogen compound frequently found in sewage. Nitrifiers can oxidize ammonium to nitrate and, together with denitrifiers, produce gaseous N-forms without large increases in sludge biomass (la Cour Jansen et al. 1997). The nitrification pathway success relies on the synergic activity and interaction of two different microbial groups: ammonia oxidizers and nitrite oxidizers. Firstly, ammonia (NH3) is oxidized by ammonia-oxidizing bacteria (AOB) or ammonia-oxidizing archaea to nitrite (), and then further oxidized by nitrite-oxidizing bacteria (NOB) to nitrate () (Bai et al. 2012). However, nitrifiers are sensitive to a large number of environmental factors and culture conditions including dissolved oxygen (DO) concentration, temperature, pH and the presence of inhibitory compounds, such as phenols. As a consequence, nitrification often becomes the rate-limiting step for nitrogen removal in wastewater treatment plants.
There are several studies characterizing nitrifying consortia, which may be sessile in activated sludge (Ni et al. 2011) or biofilms (aerated filters). Most of these studies revealed that Nitrosomonas (AOB) and Nitrospira (NOB) are the most abundant nitrifiers in wastewater treatment processes (Kindaichi et al. 2004; Waheed et al. 2013), while others members of the community belong to the green nonsulfur bacteria, Gammaproteobacteria, Cytophaga–Flavobacterium–Bacteroidetes group, Alphaproteobacteria and Verrucomicrobia (Kindaichi et al. 2004). The heterotrophic organisms associated with the nitrifying consortium assimilate dead biomass and metabolites from the autotrophic nitrifiers (Okabe et al. 2005).
Five years ago, a laboratory-scale continuous stirred tank reactor (CSTR) was started up using sludge from a wastewater plant as inoculum, ammonium (NH4+) as the sole energy source and sodium bicarbonate (NaHCO3) to maintain a high dissolved CO2 concentration. The microbial population in the nitrifying reactor has been kept under the same conditions and provides inocula for respirometric studies (Ramirez-Vargas et al. 2013). Consequently, a unique bacterial consortium has been established with the micro-organisms being planktonic or free-swimming. This unique autotrophic–heterotrophic interacting community is still unexplored and was investigated by 454 pyrosequencing of the 16S rRNA gene. The objective of this study was to determine the autotrophic–heterotrophic bacterial community structure in a nitrifying reactor maintained under the same conditions for more than 5 years using a high-throughput approach to get a deeper insight into the bacterial community. In addition, this study might help us to understand the interaction between autotrophic nitrifiers and heterotrophs in wastewater treatment plants and the factors that control it. It was hypothesized that the specific conditions in the reactor and the long-term experiment (5 years) selected for a specialized consortium that will help us understand the metabolic links between autotrophic and heterotrophic bacteria in a wastewater treatment plant.
Autotrophic mixed culture
The bacterial community structure was determined in a nitrifying reactor described by Ramirez-Vargas et al. (2013). The microbial population in the bioreactor was selected for an autotrophic nitrifying population by using ammonium as sole energy source and bicarbonate as carbon source. The culture was obtained from a wastewater treatment plant and added to a glass CSTR (0.14 m diameter, 0.56 m height and 5.5 L working volume). The CSTR was operated continuously under steady-state for 5 years to study and calculate kinetic and stoichiometric parameters through respirometric techniques. The reactor was fed with a solution containing (g/L distilled water) (NH4)2SO4, 1.73; NH4Cl, 1.40; KH2PO4, 2.73; FeCl3, 0.012; MgSO4, 0.60; NaCl, 1.00; CaCl2, 0.05; NaHCO3, 9.3 and 5.0 mL/L of a trace element solution containing (g/L) (NH4)6Mo7O24·4H2O, 0.08; ZnSO4·7H2O, 0.1; CuSO4·5H2O, 0.02; CoCl2·6H2O, 0.002; MnCl2·4H2O, 0.2. The solution contained no organic C and only C in inorganic bicarbonate form. Ammonium and bicarbonate concentrations were such that a C/N ratio of 1.8 was maintained. The mineral solution was fed with a constant flow rate of 0.69 L/d (hydraulic residence time of 8 days), using a peristaltic pump (Masterflex L/s precision, Cole-Parmer, USA). Air was supplied continuously at a flow rate of 1 vvm controlled by a mass flow controller (GFC171S, Aalborg, Denmark). The solids retention time (SRT) for our reactor was 55 days (Cervantes et al. 2006). The DO was maintained at 5.1 mg O2/L, and monitored with a polarographic DO bench meter (HI2400, Hanna Instruments). The pH was maintained at 7.5 ± 0.2 using either 1 M NaOH or 1 M H3PO4. The reactor was maintained at room temperature (21 ± 2 °C). During operation effluent biomass was settled and recycled to the reactor to increase the biomass concentration. The ammonium, nitrate and nitrite concentration was monitored every 5 days.
Extraction of total DNA
Three 10 mL sub-samples were taken from the bioreactor with a week interval between them and total DNA was extracted with enzymatic lysis using the automated QIAcube method (Qiagen, Venlo, The Netherlands). Aliquots of 2 mL of each sample were centrifuged at 12,000 × g for 5 minutes and the pellets were resuspended in 200 μL sterile distilled water. For lysis, 180 μL lysozyme (20 mg/mL) were added to each tube, followed by incubation at 37 °C for 1 hour. The samples were processed by the QIAcube-robot and eluted in a final volume of 200 μL water. The DNA-yield was quantified with a UV transilluminator (Gel Doc 2000, Bio-Rad Laboratories Inc., Carlsbad, CA, USA) after 0.8% (w/v) agarose gel electrophoresis and staining with an ethidium bromide solution (1.5%). The DNA extracted was stored at −20 °C until used for polymerase chain reaction (PCR) amplification.
Total DNA was used as a template for PCR amplification of 16S rRNA gene. Ribosomal libraries of V1–V6 regions were obtained using 10 bp barcoded primers 8–F (5′-AGA GTT TGA TCI TGG CTC A-3′) (Navarro-Noya et al. 2013) and 946–R (5′-CCG TCW ATT KCT TTG AGT T-3′). The PCR mixture (25 μL) contained 100 ng DNA with the appropriate primers at 0.5 μM; 1× PCR buffer; 2.5 mM MgCl2; dATP(deoxyadenosine triphosphate), dCTP (deoxycytidine triphosphate), dGTP (deoxyguanosine triphosphate) and dTTP (thymidine triphosphate) each at a concentration of 200 μM and 1 U of Taq DNA polymerase (Thermo Scientific, Waltham, MA, USA). The reactions were done with initial denaturation at 94 °C for 10 minutes, followed by 25 cycles of 94 °C for 1 minute, annealing at 56 °C for 1 minute and 72 °C for 1.5 minutes, followed by a final extension at 72 °C for 10 minutes. The PCR products were analyzed for size and purity with agarose gel (0.8%) electrophoresis and UV transillumination after staining with ethidium bromide.
Each DNA sample was amplified five times, and the PCR products were pooled and purified using the UltraClean™ PCR clean-up kit (MoBio, Carlsbad, CA, USA). Equal concentrations of the purified library amplicons were sent for pyrosequencing. Pyrosequencing was done with a Roche GS–FLX Plus 454 pyrosequencer (Roche, Mannheim, Germany) by Macrogen Inc. (DNA Sequencing Service, Seoul, Korea).
Analysis of pyrosequencing data
The QIIME version 1.5.0 software pipeline was used to analyze the pyrosequencing data (Caporaso et al. 2010b). Details can be found in Navarro-Noya et al. (2013). First, the poor quality reads were eliminated from the data sets. Second, operational taxonomic units (OTUs) were determined at 97% similarity level (UCLUST algorithm) (Edgar 2010). Third, chimeras were removed using the Chimera Slayer (Haas et al. 2011). Fourth, sequences were aligned against the Greengenes core set using representative sequences of each OTU with PyNAST and filtered at a 75% threshold (Caporaso et al. 2010a).
Phylogenetic and statistical analysis
The phylogenetic distribution was determined at different taxonomic levels (80% confidence threshold) by the naïve Bayesian rRNA classifier from the Ribosomal Data Project (http://rdp.cme.msu.edu/classifier/classifier.jsp) (Wang et al. 2007).
RESULTS AND DISCUSSION
Aerobic oxidation of NH3 to and of to by nitrifiers generates energy for autotrophic growth, while the microbial products derived from nitrifiers (metabolites and death cell material) provide the only organic material for heterotrophs. The nitrifying reactor was inoculated and operated for 5 years under a constant loading rate of 95.2 ± 1.5 mg -N/(L d). From the onset, nitrification was observed (Figure 1). The ammonia loading rate rapidly decreased from 95.2 mg -N/(L d) to a loading rate of 9.4 ± 2.1 mg -N/(L d). As a consequence of the nitrification activity, an increase in the nitrate discharge rate was detected (74.8 ± 2.3 mg -N/(L d)) as well as an increase in the biomass concentration (1,911.66 ± 13 mg chemical oxygen demand (COD)/L). From the data presented in Figure 1, it can be concluded that the system reached a steady state after approximately 40 days of continuous operation. As such, both AOB and NOB were active.
The efficient removal of in nitrification treatment systems relies on the interaction between two phylogenetically unrelated groups, AOB and NOB. In the nitrifying reactor, the AOB phylotypes belonged to the genus Nitrosomonas (Betaproteobacteria) with four different OTUs (11.0 ± 1.2%) (Figure 2). One of the most abundant sequences belonged to Nitrosomonas europaea and the other to Nitrosomonas mobilis. The NOB phylotypes belonged to the genus Nitrobacter (Alphaproteobacteria) with two different OTUs (9.3 ± 0.6%). The most abundant sequence resembled Nitrobacter winogradskyi. Ammonia-oxidizing bacteria in activated sludge from a municipal wastewater treatment plant in Hong Kong and in a carbon-limited autotrophic nitrifying biofilm were also dominated by phylotypes that belonged mainly to Nitrosomonas, but Nitrospira was the most abundant NOB (Kindaichi et al. 2004; Feng et al. 2012; Yu & Zhang 2012). The presence of Nitrobacter rather than Nitrospira in the reactor might be determined by the DO and/or the concentration of (Huang et al. 2010). Nitrospira spp. have shown high affinity for oxygen and nitrite, enabling their dominance in low-oxygen regions over Nitrobacter spp. (Downing & Nerenberg 2008). Liu & Wang (2013a, b) found that in long-term low DO conditions, the number of Nitrospira-like bacteria increased, while that of Nitrobacter-like bacteria decreased. The reactor was well aerated, so Nitrobacter spp. should be favored. Bartosch et al. (1999) stated that Nitrobacter cells prefer microenvironments with higher concentrations while Nitrospira-like cells dominate when the concentration is lower. Nogueira & Melo (2006) reported that Nitrobacter as an r-strategist with rapid growth dominates when resources () are abundant, while Nitrospira, which grows more slowly (K-strategist), dominates when resources are limited. Our reactor is resource rich as large amounts of are supplied, so Nitrobacter spp. are favored as the available is constantly high.
Apart from defining the Nitrospira–Nitrobacter ratio, the oxygen concentration affects also the relative abundance of both groups. Experiments in mixed continuous cultures of Nitrosomonas europaea and Nitrobacter winogradskyi showed that under oxygen-limiting conditions, the Nitrosomonas population increased in relation to the Nitrobacter winogradskyi cells, mainly because they are better competitors in these conditions (Laanbroek & Gerards 1993). In the nitrifying reactor with non-limiting oxygen conditions, both genera showed a similar relative abundance, i.e. Nitrosomonas spp. 11.1% and Nitrobacter spp. 9.4%. As a result, the amount of -N in the influent was similar to the -N in the effluent, suggesting a 1:1 coupling between AOB and NOB in the reactor.
The SRT for our reactor was 55 days. Liu & Wang (2014) showed that with a short SRT ammonia oxidation was favored over nitrite oxidation and with a long SRT nitrite oxidation matched or surpassed ammonia oxidation. Our results showed no ammonia or nitrite accumulation in the reactor, which indicates that our nitrifying reactor was always working at a long and correct SRT.
The ratio of total nitrifiers (AOB plus NOB) to all heterotrophs was 1:4 (Figure 2). For autotrophic biofilms of approximately 3 months in age, Kindaichi et al. (2004) reported a value of 1:1, while Okabe et al. (2005) reported values of 2:1. It is difficult to know why the ratio of total nitrifiers to all heterotrophs was much lower in this study than in the reported studies, but several factors are different which might have affected this ratio. The lower percentages of nitrifiers in our study compared to the other studies can be due to the fact that the reactor conditions were different and the bacterial population was planktonic and not fixed in a biofilm. For instance, Ni et al. (2011) reported that heterotrophic growth was higher in nitrifying biofilm (30–50%) and granules (30%) than in nitrifying sludge (15%). Okabe et al. (2005) had to disturb the spatial organization of the microbial community in the biofilm as homogenization was necessary for quantitative MAR-FISH (microautoradiography–fluorescent in situ hybridization) analysis. They stated that this disturbance might have affected metabolic activity, the ratio of total nitrifiers to all heterotrophs and the bacterial community structure. In addition, the bacterial consortium in the nitrifying reactor was much older in this study than in the other studies, i.e. 5 years in this study and only 3 months in the studies by Okabe et al. (2005) and Kindaichi et al. (2004), so that a larger heterotrophic population might have developed. The coexistence of a high level of heterotrophs with nitrifiers has been found often in autotrophic nitrifying biofilms cultured without an external organic carbon supply (e.g. Rittmann et al. 1994; Okabe et al. 2002). Autotrophic nitrifiers and heterotrophs interacting through the exchange of organic matter; the autotrophic nitrifiers reduce inorganic C to organic C in cell mass, and produce and release soluble microbial products into solution to maintain a heterotrophic population.
Phylotypes in the autotrophic mixed reactor belonged to 10 different phyla (Figure 2(a)). The most abundant bacterial phylum was Proteobacteria (relative abundance 62.8 ± 1.9%) followed by Bacteroidetes (30.8 ± 2.4%). These two phyla often dominate the bacterial community in activated sludge of wastewater treatment plants (Feng et al. 2012; Ibarbalz et al. 2013). Within the Proteobacteria, Alphaproteobacteria were the most abundant (42.3 ± 2.8%) followed by Betaproteobacteria (11.1 ± 1.2%) and Gammaproteobacteria (9.4 ± 2.2%) (Figure 2(a)). Members of the Alpha- and Gammaproteobacteria use preferentially low-molecular-weight organic material, i.e. acetic acid and amino acids (Kindaichi et al. 2004; Okabe et al. 2005), so they are well adapted to thrive in low-organic-load environments, such as rivers and marine environments (Kenzaka et al. 1998), autotrophic nitrifying biofilms (Kindaichi et al. 2004; Okabe et al. 2005) and the reactor used in this study. Alpha- and Gammaproteobacteria are involved in the mineralization of microbial products, but not directly in the decomposition of cell-wall material, i.e. N-acetylglucosamine-6-phosphate (NAG) (Okabe et al. 2005). Betaproteobacteria are often dominant in heavily polluted rivers (Brummer et al. 2000) and activated sludge systems with large amounts of organic material (Wagner & Loy 2002). Okabe et al. (2005) reported that the Betaproteobacteria did not participate in the mineralization of organic material derived from nitrifying bacteria; so this suggested that they were less competitive in ecosystems with low concentrations of organic material. In the 3-month-old autotrophic nitrifying biofilm, still 6% of the sequences belonged to Betaproteobacteria (Okabe et al. 2005), but only 0.08% (not considering the phylotypes belonging to the genus Nitrosomonas) in the 5-year-old nitrifying reactor.
The genus Mesorhizobium (23.3 ± 2.5%) was the most abundant in the nitrifying reactor and the sequences contained three different OTUs, but phylogenetic analysis did not reveal a lower taxonomic level. Phylotypes belonging to the genus Mesorhizobium were found in a biofilm attached to the porous support of an aerobically operated two-stage rectangular packed-bed biofilm reactor treating sulfonated azo dyes (de los Cobos-Vasconcelos et al. 2012). Different strains belonging to the genus Mesorhizobium (e.g. M. cicero biovar biserrulae WSM1271, M. loti MAFF303099, M. opportunistum WSM2075 and M. australicum WSM2073) contain the gene nagA that encodes for the enzyme N-acetylglucosamine-6-phosphate deacetylase and the gene nagB that encodes for the enzyme glucosamine-6-phosphate deaminase (Yang et al. 2006). Strains of the Mesorhizobium also contain the nirK gene (nitrite reductase), but not the norBDQ (nitric reductase) and nosZ genes (nitrous oxide reductase) (Monza et al. 2006). The presence of these genes might favor the phylotypes belonging to Mesorhizobium in the nitrifying reactor, although other metabolic characteristics might explain their high relative abundance.
Luteimonas was the third most abundant genus (6.8 ± 1.9%) in this study, not considering the two nitrifying genera (Figure 2(b)). Interestingly, Luteimonas was first isolated from an ammonia-supplied biofilter (Finkmann et al. 2000). There is yet no evidence that strains belonging to the genus Luteimonas can degrade NAG, but they can use as electron acceptor just as strains belonging to the genus Mesorhizobium can (Finkmann et al. 2000). The high relative abundance of phylotypes belonging to the Bacteroidetes in the reactor can be attributed to their capacity to use organic material of high molecular mass, such as NAG (Cottrell & Kirchman 2000). Kindaichi et al. (2004) showed that only members of Chloroflexi and Bacteroidetes metabolized NAG, i.e. a product of cell wall decay. Nearly all phylotypes belonging to the Bacteroidetes belonged to the Flavobacteriia (29.9 ± 2.4%) and only 0.9 ± 0.2% belonged to the Sphingobacteriia. The Flavobacteriia were the second most important group in the 5-year-old nitrifying reactor. They are capable of metabolizing cell wall material, extracellular polymeric substances produced in large quantities by nitrifying bacteria and secondary metabolites of the decayed biomass (Okabe et al. 2005; Bernardet & Bowman 2006). Phylogenetic analysis of the OTUs indicated that phylotypes belonging to the Flavobacteriaceae contained three different OTUs (Figure 2(c)). The most abundant OTU was affiliated in a maximum likelihood phylogenetic tree with Aequorivita antartica (data not shown).
The relative abundance of the Chloroflexi was low in this study (0.23 ± 0.05%), although Okabe et al. (2005) stated that they participate in the degradation of dead bacterial cells in the autotrophic nitrifying biofilms studied. Reactor conditions might have reduced the relative abundance of the Chloroflexi or the capacity of the Bacteroidetes to degrade various refractory biomacromolecules, e.g. cellulose, chitin and proteins (Okabe et al. 2005; Fernandez-Gomez et al. 2013).
Although phylotypes belonging to the Actinobacteria have often been reported as the third most important phylum in activated sludge of wastewater treatment plants (Ibarbalz et al. 2013), their relative abundance in the nitrifying reactor studied was only 2.1%. The relative abundance of phylotypes belonging to the Firmicutes was also low in this study (0.11%), although much higher values have been reported in activated sludge of wastewater treatment plants (>40%) (Ibarbalz et al. 2013). Firmicutes and Actinobacteria respond readily to easily decomposable organic material, which can often be found in large amounts in activated sludge (Placella et al. 2012), but the low-organic-load nitrifier reactor will not have favored them. Apart from the phylum Deinococcus-Thermus (1.09), the relative abundance of all other phyla was <1%.
It was found that the ammonia oxidizers in the nitrifying reactor belonged to the genus Nitrosomonas while the nitrite oxidizers belonged to the genus Nitrobacter. Those nitrifiers sustained a broad population of heterotrophs (>70 different bacterial groups at the genus level) dominated by phylotypes belonging to Alpha- and Gammaproteobacteria and Bacteroidetes. The dominance of these proteobacterial groups and the absence of the Betaproteobacteria (not considering the AOB%) seems to indicate that the first thrived in low-organic-load environments while the latter are not well adapted to these conditions. Mesorhizobium was the most abundant genus in the nitrifying reactor and different strains belonging to this group contain the genes nagA and nagB that encode for enzymes involved in the degradation of NAG formed after depolymerization of cell wall peptidoglycan. Luteimonas, first isolated from an ammonium-supplied biofilter, was also an abundant genus with no known capacity to metabolize NAG, but with the capacity (also found in Mesorhizobium) to use nitrite as electron acceptor. The Flavobacteriaceae, known to metabolize cell wall material, extracellular polymeric substances produced in large quantities by nitrifying bacteria and secondary metabolites of the decayed biomass, was another dominant group of bacteria in the nitrifying reactor.
Authors Rocio Ramirez-Vargas and Nancy Serrano-Silva contributed equally to this work. This research was funded by Cinvestav (Mexico). Y. N.-N. received a postdoctoral grant from ABACUS ‘Consejo Nacional de Ciencia y Tecnología, México’ (CONACyT) and R. R.-V. and N. S.-S. a doctoral grant from CONACyT.