Study Of Membrane-Aerated Biofilm Reactor For Single-Stage Autotrophic Nitrogen Removal

Removing nitrogen components from wastewater is an important issue nowadays since these components can cause eutrophication in receiving water. However conventional biologial nitrogen removal processes exit some problems such as system complex, large footprint, high operating costs, and ect. Recently, a novel and promising microbial process combining the Anammox reaction with partial nitrification in one reactor, entitled single-stage autotrophic nitrogen removal has been developed. This process would lead to an important reduction of operational costs compared to coventional nitrification-denitrification processes.In this study, a novel membrane-aerated biofilm reactor(MABR), equipped with non-woven materials support around the microporous carbon tube membrane, was developed for single-stage autotrophic nitrogen removal based on partial nitrification and anaerobic ammonia oxidation(ANAMMOX). The MABR was start-up via sequential addition of two types of seeding sludge: aerobic nitrifying and ANAMMOX sludge. Initially, the reactor was inoculated with nitrifying biomass. By decreasing the air pressure in the lumen of carbon tube step by step, one can obtain stable nitrite accumulation. Subsequently, the reactor was reinoculated with Anammox biomass enriched by a upflow column reactor packed with nonwoven fabrics carrier, to construct a quite stable biofilm system for single-stage autotrophic nitrogen removal. This study was performed as follows:(1) The upflow column reactor packed with nonwoven fabrics carrier was used for enrichment of incubated ANAMMOX sludge. After 101 d operation, the ANAMMOX sludge concentrations in reactor was enhanced form 470 mg L^-1 to 3118.4 mg L^-1. In the stable stage of operation, the average TN volumetric loading rate was 818.3 mg L^-1, the maximum TN removal efficiency was 63.8 % with the influent NH₄⁺-N concentration of 200-250 mg L^-1. The morphology of the cultivated ANAMMOX sludge was observed using scanning electron microcope. The photographs showed that the typial cauliflower-like aggregates consisted of mostly spherical in shape in the obtained culture.(2) The molecular biological techniques PCR, clone, FISH and real-time PCR were utilized to study prinimilarily the microorganism in the upflow column reactor. The Planctomycetales clone results showed that there are two kinds of bacteria in the culture: one had＞98 % similar with the KSU-1 sequence(Genbank ID: AB057453.1), the other had 100 % similar with the Uncultured Planctomycetes bacterium gene for 16S rRNA(Genbank ID: AB176696.1). Fluorescene in situ hybridization (FISH) analysis using 16S rRNA targeting oligonucleotide probes PLA46 showed that the dominant population developed in the upflow column reactor was hybridized with PLA46 gene probes. It means that the cultivated biomass in the reactor was classified in the group of Planctomycetales bacteria. The results of real-time PCR showed that there are 6.32×10⁴ bacteria 16S rDNA gene copy and 1.58×10⁴ Planctomycetales bacteria 16S rDNA gene copy in the 1μl culture. The Planctomycetales bacteria occupied 45-60 % of the bacteria total amount.(3) The operation of the MABR for single-stage autotrophic nitrogen removal was successful during the tested period. During the test period, the maximal nitrogen removal was 0.766 kg N m^-3 d^-1 with a volumetric loading of 0.96 kg N m⁽-30 d^-1, intra-membrane air pressure of 0.015 MPa, reactor temperature of 35℃and pH of 7.8.(4) Based on these aerobic and anaerobic activity measurements, it was confirmed that the partial nitrification occurred in the regions near the membrane-biofilm interface, and the Anammox occurred in the regions near the biofilm-liquid interface.(5) Fluorescene in situ hybridization (FISH) analysis using 16S rRNA targeting oligonucleotide probes EUB338 plus, NSO190, AMX820 and PLA46 showed that theexistence of two visible active layers in experimental MAB. One is the partial nitrifying layer located in the region of oxygen-rich membrane-biofilm interface, dominated by NSO190-positive AOB. The other is the Anammox active layer located in the region of anoxic liquid-biofilm interface, dominated by PLA46 and AMX820-positive Anammox microorganisms. As a result of this study, the AOB as well as Anammox bacteria were present and active in experimental MABR, and the cooperation between AOB and Anammox bacteria was considered to be responsible for single-stage autotrophic nitrogen removal.(6) The nested PCR-DGGE was performed to compare 16S rDNA fragment profiles of AOB community composition of membrane-aerated biofilm in different operation stage. The DNA band profiles of AOB varied at different sampling times, suggesting that a step by step decrease of oxygen concentration by regulations of pressure in the lumen of the MABR caused a change in the AOB community composition of the inoculated sludge.The dominant AOB community was not very complicated at the steady autotrophic nitrogen removal stage of MABR. Sample of day 110 showed three strongly stained bands (A, B and C) which were excised from gels and amplified by PCR, and the nucleotide sequences were determined. The obtainable major bands of AOB community showed high similarity (＞99% similarity)to the genus Nitrosomonas affiliated with the beta subclass of the class proteobacteria. On the other hand, nested PCR-DGGE was also performed to compare 16S rDNA fragment profiles of Anammox community composition of re-inoculated sludge and MABR system at days 65 and 110. Despite the differences in sampling times, samples from day 60 and 110 of MABR revealed similar DGGE profiles, suggesting similar community composition at these stages of system performance. Sample of day 110 showed two strongly stained bands (D and E) which were most similar (＞97% similarity) to the uncultured Planctomycete bacterium affiliated with the deep-branching class Planctomycetes.