Membrane bioreactors (MBRs) have the ability to completely retain biomass and are thus suitable for slowly growing anammox bacteria. In the present study, an anammox MBR was operated to investigate whether the anammox activity would remain stable at low temperature, without anammox biomass washout. The maximum nitrogen removal rates were 6.7 and 1.1 g-N L−1 day−1 at 35 °C and 15 °C, respectively. Fluorescence in situ hybridization and 16S rRNA-based phylogenetic analysis revealed no change in the predominant anammox species with temperature because of the complete retention of anammox biomass in the MBR. These results indicate that the predominant anammox bacteria in the MBR cannot adapt to a low temperature during short-term operation. Conversely, anammox activity recovered rapidly after restoring the temperature from the lower value to the optimal temperature (35 °C). The rapid recovery of anammox activity is a distinct advantage of using an MBR anammox reactor.
Nitrogen removal during wastewater treatment is essential for the balanced development of society through environmental sustainability. Anaerobic ammonium oxidation (anammox) is a biological process mediated by anammox bacteria belonging to the Planctomycetes-like bacteria (Strous et al. 1999a). The anammox process is promising as a cost-effective method for nitrogen removal from wastewater because it requires no aeration and no added carbon source. However, anammox bacteria are known for their very slow growth so the anammox process requires long start-up periods, meaning that sufficient biomass must be maintained in the system to sustain effective operation.
Many researchers have described the physiological characteristics of anammox bacteria. The optimum temperature range of ‘Candidatus Brocadia anammoxidans', ‘Candidatus Kuenenia stuttgartiensis', and ‘Candidatus Brocadia sinica’ were reported as 20–43 °C (Strous et al. 1999b), 25–37 °C (Egli et al. 2011), and 25–45 °C (Oshiki et al. 2011), respectively. For the application of the anammox process to real wastewater treatment, several researchers have referred to the influence of lower temperatures on nitrogen removal performance (Sánchez Guillén et al. 2014; Lackner et al. in press). However, different conclusions were reached regarding the adaptation of anammox bacteria to lower temperatures. For example, several studies reported that anammox bacteria could adapt to a low temperature (Hu et al. 2013; Taotao et al. 2015). Conversely, Dosta et al. reported that a low temperature caused irreversible inhibition of anammox activity through inhibition caused by the accumulation of nitrite resulting from the lower activity at the lower temperature (Dosta et al. 2008). Ma et al. also reported that the nitrogen removal rate decreased when the temperature decreased, although sufficient nitrogen removal performance was maintained (Ma et al. 2013). Results from marine anammox species have also been reported, in which each marine anammox bacterial species had an intrinsic optimal temperature range (Awata et al. 2012). Notably, marine anammox bacteria tend to favour lower temperatures than freshwater species (van de Vossenberg et al. 2008; Awata et al. 2013).
When anammox biomass is washed out because of lower or fluctuating temperature, a long operation period would be needed to recover the nitrogen removal performance. We therefore hypothesized that membrane bioreactors (MBRs) would have benefits for the recovery of nitrogen removal performance because they completely retain biomass within the reactor. Anammox MBRs were successfully operated to investigate the characteristics of anammox bacteria (van der Star et al. 2008; Oshiki et al. 2013). However, fundamental knowledge about the operation of anammox MBRs at different temperatures is currently missing. In this study, an anammox MBR was operated at different temperatures to confirm whether reactor performance could remain stable at lower temperatures, without anammox biomass washout.
MATERIALS AND METHODS
|Period (day)||Temperature (°C)||Influent NH4+ (mg-N L−1)||Influent NO2− (mg-N L−1)|
|Period (day)||Temperature (°C)||Influent NH4+ (mg-N L−1)||Influent NO2− (mg-N L−1)|
A synthetic nutrient medium was used, containing 3.5–18 mM (NH4)2SO4, 5–24 mM NaNO2, 1.0 mM KHCO3, 0.2 mM KH2PO4, 1.2 mM MgSO4·7H2O, 1.2 mM CaCl2·2H2O, and 1 mL of trace element solutions I and II, as described by van de Graaf et al. (1996). The pH was adjusted to 6.5–7.5 using HCl or NaOH. The medium was flushed with N2 gas for at least 1 hour before adding the nutrients to achieve a dissolved oxygen concentration below 0.5 mg L−1.
The concentrations of , , and were determined using ion-exchange chromatography (HPLC 20A, Shimadzu Co., Kyoto, Japan) with a Shim-pack IC-C4 (Shimadzu) or a Shodex Asahipak NH2P-50 4D anion column (Showa Denko, Tokyo, Japan) and a conductivity detector (CDD-10Avp, Shimadzu) or UV-VIS detector (SPD-10A, Shimadzu) after filtration of the samples through 0.2-μm pore-size cellulose acetate membranes (Advantec Co., Tokyo, Japan) (Awata et al. 2015). The total nitrogen loading and removal rates were calculated based on the concentrations of , , and , and the hydraulic retention time.
Total DNA was extracted from the MBR at the end of Phase I (day 28) and Phase II (day 84) using the Fast DNA spin kit for soil (MP Biomedicals, Irvine, CA, USA) according to the manufacturer's instructions. To construct the clone libraries (Library-35 °C and Library-15 °C), 16S rRNA gene fragments were amplified using the Planctomycetales-specific primer sets Pla46f (Neef et al. 1998) and 1390r (Zheng et al. 1996). The PCR (polymerase chain reaction) conditions were as follows: 4 minutes of initial denaturation at 94 °C, followed by 30 cycles of 45 s at 94 °C, 50 s at 58 °C, and 3 min at 72 °C. The final extension was performed for 10 minutes at 72 °C. PCR products were confirmed using a 1% (w/v) agarose gel and were purified using the QIAquick PCR Purification Kit (Qiagen, Hilden, Germany). The purified PCR products were ligated into a pCR-XL-TOPO vector and transformed into One Shot Escherichia coli cells, following the manufacturer's instructions (TOPO XL PCR cloning kit; Invitrogen, Carlsbad, CA, USA). Sequences with 97% or greater similarity were grouped into operational taxonomic units (OTUs) using the ARB software (Ludwig et al. 2004). The phylogenetic tree was constructed using neighbour-joining (distant matrix) with Jukes–Cantor correction, maximum parsimony (Phylip DNAPARS), and maximum likelihood (RAxML) with GTR Gamma model methods in the ARB software, with a database SSU Ref NR release 119 (Pruesse et al. 2007). Bootstrap resampling analysis of 1,000 replicates was performed to estimate the confidence of tree topologies. The sequence data of the partial 16S rRNA gene obtained from the MBR were deposited in the GenBank/EMBL/DDBJ databases under accession numbers LC053452 and LC053453.
Fluorescence in situ hybridization analysis
Biomass samples were obtained from Phase I (day 28) and Phase II (day 84) to compare the microbial community composition of anammox bacteria, and were fixed in a 4% paraformaldehyde solution for 8 hours at 4 °C. In situ hybridization was conducted according to the procedure described by Okabe et al. (1999), and a model Axioimager M1 epifluorescence microscope (Carl Zeiss, Oberkochen, Germany) was used for the observation. The 16S rRNA-targeted oligonucleotide probes used in this study were EUBmix, which was composed of EUB338 (Amann et al. 1990), EUB338II, and EUB338III (Daims et al. 1999), and Amx820 (Schmid et al. 2001). The probes were labelled with Cy3 or Alexa Fluor 488 at the 5′ end. For the quantitative determination of microbial composition in the granules, the surface fraction of the specific probe-hybridized cell area and EUBmix probe-hybridized cell area were determined after gentle homogenization (Kindaichi et al. 2004). The average fraction was determined from 16 representative fluorescent images using ImageJ software (Collins 2007).
RESULTS AND DISCUSSION
Effects of temperature on nitrogen removal
Microbial community structure
An anammox MBR was operated to investigate the influence of temperature on nitrogen removal performance and the microbial community structure of anammox MBR. The nitrogen removal rate decreased with decreasing operational temperature, but the community composition and predominant species of anammox bacteria did not change significantly. No adaptation of anammox bacteria to the lower temperature was observed during short-term operation in the anammox MBR. However, the nitrogen removal performance of the MBR rapidly recovered with increasing temperature, which is one of the advantages of using the MBR. In future work, we are interested in confirming whether the population and microbial community of anammox bacteria in an MBR may change during long-term operation at lower temperatures.
This research was partially supported by a Grant-in-Aid for Scientific Research (C) from the Ministry of Education, Culture, Sports, Science and Technology by the Japan Society for the Promotion of Science (JSPS).