Characterization of an Autotrophic Nitrogen-Removing Biofilm from a Highly Loaded Lab-Scale Rotating Biological Contactor

Abstract

In this study, a lab-scale rotating biological contactor (RBC) treating a synthetic NH 4 ؉ wastewater devoid of organic carbon and showing high N losses was examined for several important physiological and microbial characteristics. The RBC biofilm removed 89% ؎ 5% of the influent N at the highest surface load of approximately 8.3 g of N m؊2 day ؊1 , with N 2 as the main end product. In batch tests, the RBC biomass showed good aerobic and anoxic ammonium oxidation (147.8 ؎ 7.6 and 76.5 ؎ 6.4 mg of NH 4 ؉ -N g of volatile suspended solids [VSS] ؊1 day ؊1 , respectively) and almost no nitrite oxidation (< 1 mg of N g of VSS ؊1 day ؊1 ). The diversity of aerobic ammonia-oxidizing bacteria (AAOB) and planctomycetes in the biofilm was characterized by cloning and sequencing of PCR-amplified partial 16S rRNA genes. Phylogenetic analysis of the clones revealed that the AAOB community was fairly homogeneous and was dominated by Nitrosomonas-like species. Close relatives of the known anaerobic ammonia-oxidizing bacterium (AnAOB) Kuenenia stuttgartiensis dominated the planctomycete community and were most probably responsible for anoxic ammonium oxidation in the RBC. Use of a less specific planctomycete primer set, not amplifying the AnAOB, showed a high diversity among other planctomycetes, with representatives of all known groups present in the biofilm. The spatial organization of the biofilm was characterized using fluorescence in situ hybridization (FISH) with confocal scanning laser microscopy (CSLM). The latter showed that AAOB occurred side by side with putative AnAOB (cells hybridizing with probe PLA46 and AMX820/KST1275) throughout the biofilm, while other planctomycetes hybridizing with probe PLA886 (not detecting the known AnAOB) were present as very conspicuous spherical structures. This study reveals that long-term operation of a lab-scale RBC on a synthetic NH 4 ؉ wastewater devoid of organic carbon yields a stable biofilm in which two bacterial groups, thought to be jointly responsible for the high autotrophic N removal, occur side by side throughout the biofilm.Sustainable wastewater treatment systems are being developed that minimize energy consumption, CO 2 emission, and sludge production. However, these systems typically yield effluents rich in ammonium-nitrogen (NH 4 ϩ -N) and poor in biodegradable organic carbon, thereby making them less suitable for biological N removal through the conventional nitrification-denitrification sequence.Different N removal processes that could be successfully integrated in a sustainable wastewater treatment system are being studied. The Sharon process (single-reactor high-activity ammonium removal over nitrite) (15) uses the principle that at higher temperatures (30 to 35°C), pH 7 to 8, and a cell residence time of 1 day, aerobic ammonia-oxidizing bacteria (AAOB) are able to maintain themselves in the system while nitrite-oxidizing bacteria (NOB) are washed out. Given the reaction stoichiometry of the two groups of nitrifying bacteria (equations 1 and 2), this proces...

Nitrogen removal through different pathways in an aged refuse bioreactor treating mature landfill leachate

“…qPCR assays were performed using SYBR Green I and conducted in FTC2000 fluorescence real-time PCR system (Canada). Primer pairs included Bacteria1055f (5′-ATGGCTGTCGTCAGCT-3′) and Bacteria1392r (5′-AC GGGCGGTGTGTAC-3′) for total bacterial 16S rRNA genes (Egli et al 2001), amoA-1 F (5′-GGGGTTTCTACTGGTG GT-3′) and amoA-2R (5′-CCCCTCKGSAAAGCCTTCTTC-3′) for AOB amoA genes (Pynaert et al 2003), and anammox-1 (5′-GGATTAGGCATGCAAGTC-3′) and anammox-2 (5′-TCTGTATTACCGCGGCT-3′) for anammox bacteria 16S rRNA genes (Tal et al 2006;Hu et al 2010). The 50-μL PCR reaction contained 25 μL of 2× PCR buffer, 1 μL of each primer (25 μM), 0.5 μL of SYBR Green I fluorescent dye, 2 μL of DNA template, and DEPC water.…”

“…Anaerobic ammonium-oxidizing (anammox) bacteria are able to oxidize ammonium with nitrite or nitrate as electron acceptor under anoxic conditions (Strous et al 1999;Pynaert et al 2003;Tal et al 2005). While nitrogen removal in a landfill bioreactor was mostly thought as nitrification and denitrification, anammox is seldom reported because there is no convenient condition to allow anammox to occur (Price et al 2003).…”

Potential roles of anaerobic ammonium and methane oxidation in the nitrogen cycle of wetland ecosystems

“…In oxygen-limited environments, the AOB would oxidize ammonium to nitrite and keep the oxygen concentration low, while anammox bacteria would convert the produced nitrite and the remaining ammonium to dinitrogen gas. Such conditions have been established in many different reactor systems (Third et al 2001;Pynaert et al 2003). The NOB cannot compete in obtaining oxygen with AOB and also cannot compete in obtaining nitrite with anammox bacteria.…”

Start-up of Autotrophic Nitrogen Removal Reactors via Sequential Biocatalyst Addition

“…At the end of the experiment (day 136), the biofilm was gently removed from the carrier with a fine brush while washing with distilled water until no biomass was visibly present. Subsequently, the amount of TSS/VSS in the reactor was determined, and specific aerobic and anoxic batch activity measurements were performed as described previously (22). Additional samples were taken for microbial characterization.…”