Study On Nitrogen Removal Performance By Immobilized Anammox Enrichment Culture

Anaerobic ammonium oxidation (ANAMMOX) has been put forward recently as a promising alternative to treat ammonium rich wastewaters. However, the ANAMMOX bacteria have a low growth rate and the biomass yield is low. For these reason, maintaining a sufficient amount of ANAMMOX sludge in a reactor is the main factor in the development of a stable and high-rate ANAMMOX system. Fortunately, immobilization is an efficient method to prevent biomass from being washed out and to promote hyper-concentrated cultures. Compared with natural microorganism technology, immobilization of microorganism technology has the advantages such as high cell density, strong endurance of toxicity, easy separation of produce, low operating expenses, simple maintenance management and little residual sludge etc. In order to promote the application of ANAMMOX process, it was necessary to research immobilized ANAMMOX process.In this research, the ANAMMOX sludge was cultured by the anaerobic sequencing batch reactor (ASBR). The best entrapped support was obtained by comparing immobilization operation, nitrogen removal ratio and cost of entrapped materials etc. and the best operating conditions was demonstrated by orthogonal experiment. The functional microbial group was identified by making specific 16srDNA clone library. In addition, the effect of hydraulic retention time (HRT), pH, and temperature on immobilized beads were investigated. Then, with immobilized ANAMMOX beads as seed, the start-up, operation, impact factors of ANAMMOX ASBR were investigated in detail.ANAMMOX ASBR was successfully started up within 126d, with aerobic nitrifying sludge as inoculum. When the ANAMMOX ASBR was run at stable state, the volumetric load rate was 0.53 kg(TN)/m³.d, and the average removal efficiency of total nitrogen was 83%. SEM and TEM showed that the typical ANAMMOX bacteria exist in the sludge.Four materials, sodium carboxymethylcellulose (Na-CMC), sodium alginate (SA), polyvinyl alcohol (PVA), and mixed liquor of polyvinyl alcohol and sodium alginate (PVA-SA), were prepared as carriers for entrapping ANAMMOX sludge. The best entrapment support PVA-SA was selected by comparing ANAMMOX efficiency, the physical characters, immobilization operation, and costs of entrapment materials and so on. Moreover, PVA-SA gel entrapment orthogonal test was performed, consequently, the PVA-SA immobilized live ANAMMOX beads were prepared.The physicochemical characteristics of PVA-SA immobilized ANAMMOX beads were investigated. The results indicated that the PVA-SA immobilized ANAMMOX beads had preferable mechanical strength, surface structure and diffusibility. By means of scanning electron microscopy, transmission electron microscopy and digital photography, the surface structure of PVA-SA immobilized bead is loose and finely porous which facilitates diffusion of the nitrogen,and the ANAMMOX bacteria were confirmed in gel beads. Then, the effect of hydraulic retention time (HRT), pH, and temperature on immobilized biomass were investigated. The effect of pH and temperature on the ANAMMOX process was evidently weakened in PVA-SA immobilized gel beads and the effect of HRT on the ANAMMOX reaction was reversely significant, which could be the key parameter of the immobilized ANAMMOX system. Stable operation could be achieved in an ASBR, which proved that gel immobilization was an excellent method to maintain the ANAMMOX biomass.PCR amplification, cloning, and phylogenetic analysis of the 16S rRNA genes were performed to identify the ANAMMOX bacteria immobilized in the gel bead culture. The results demonstrated that there are one ANAMMOX bacterial species existing in the gel beads (GenBank accession numbers EU661861). Phylogenetic analysis based on 16S rRNA sequences revealed that these bacteria were groups of distantly relative microorganisms from the Planctomycete community, had a common node with the recently described anaerobic ammonia-oxidizing group KSU-1(AB057453) of the Planctomycetales. The test results indicated that the efficiency of ANAMMOX has linear relationship with the growth of biomass, it showed the sufficient ANAMMOX biomass maintain in reactor is necessary. The kinetic characteristics of anaerobic ammonia oxidation was studied, The cell yield was 0.0645mgVS (mgNH₄⁺)^-1 and the cell decay coefficient was 0.0452mgVS (g VS·d)^-1, the Mathematical expression wasμ= 0.0645U - 0.0452 (r2=0.9641).Finally, the ASBR was performed using ANAMMOX bacteria entrapped in a gel carrier. There appeared to be no lag phase in activity of the ANAMMOX bacteria, because the seed sludge was ANAMMOX enrichment, nitrogen removal quickly increased, and the removal ratios of ammonium and nitrite by gel beads were 81.3% and 76.5% until day 10, respectively. Although nitrogen loading increased in the influent, nitrite and ammonium concentration in the effluent decreased to below 15 mg/L and 10 mg/L after 30 day, the nitrogen conversion rate continuously increased and reached an average of 0.58 kg N/m³ per day after day 60. The effect of pH and substrate shocks on the performance of ANAMMOX reactor was investigated. Whether the reactor was affected by substrate or pH shocks, the performance was considerably affected. Comparing with substrate shocks, the reactor was more sensitive to pH shocks. The performance was able to recover from the disturbances at all the tested shocks within 5-11 days. The recovering velocity was relatively stable, which did not increase with the intensity of the shocks. But the reactor was easier to recover from high pH shocks than low high pH shocks. The organic carbon substrate test demonstrated that the organic matter could inhibit the ANAMMOX, under organic carbon condition, the heterotrophic denitrification was enhanced, and the nitrate nitrogen produced from ANAMMOX reaction could be removed.From the above results, it is believed that gel entrapment was supposed to be a highly effective technique for immobilized ANAMMOX bacteria. The immobilized ANAMMOX can bring great economic benefits due to easy separation and maintenance of the ANAMMOX bacteria in the reactor. The results proved the feasibility and good prospect of immobilized ANAMMOX, and laid a theoretical foundation for ANAMMOX process based on immobilization technique.