N-methyl pyrrolidone manufacturing wastewater as the electron donor for denitrification: From bench to pilot scale.

Actual wastewater generated from N-methylpyrrolidone (NMP) manufacture was used as electron donor for tertiary denitrification. The organic components of NMP wastewater were mainly NMP and monomethylamine (CH3NH2), and their biodegradation released ammonium that was nitrified to nitrate that also had to be denitrified. Bench-scale experiments documented that alternating denitrification and nitrification realized effective total­nitrogen removal. Ammonium released from NMP was nitrified in the aerobic reactor and then denitrified when actual NMP wastewater was used as the electron donor for endogenous and exogenous nitrate. Whereas TN and NMP removals occurred in the denitrification step, dissolved organic carbon (DOC) and CH3NH2 removals occurred in the denitrification and nitrification stages. The genera Thauera and Paracoccus were important for NMP biodegradation and denitrification in the denitrification reactor; in the nitrification stage, Amaricoccus and Sphingobium played key roles for biodegrading intermediates of NMP, while Nitrospira was responsible for NH4+ oxidation to NO3-. Pilot-scale demonstration was achieved in a two-stage vertical baffled bioreactor (VBBR) in which total­nitrogen removal was realized sequential anoxic-oxic treatment without biomass recycle. Although the bench-scale reactors and the VBBR had different configurations, both effectively removed total nitrogen through the same mechanisms. Thus, an N-containing organic compound in an industrial wastewater could be used to drive total-N removal in a tertiary-treatment scenario.

The roles of Rhodococcus ruber in denitrification with quinoline as the electron donor.

Denitrification is an important step in domestic wastewater treatment, but providing bioavailable electron donors is an expense. However, some industrial wastewaters contain organic compounds that could be a no-cost or low-cost electron donor, because they otherwise must be treated separately. In this work, quinoline was used as an electron donor to drive denitrification through bioaugmentation with Rhodococcus ruber, which is able to biodegrade quinoline. When quinoline-acclimated biomass (QAB) was used for denitrification, addition of R. ruber accelerated biodegradation of quinoline and its first mono-oxygenation intermediate (2-hydroxyquinoline). Although R. ruber was not directly active in denitrification, its biodegradation of quinoline and 2-hydroxyquinoline supplied products that other bacteria used to respire nitrate. In contrast, glucose-acclimated biomass (GAB) could not achieve effective denitrification with quinoline, whether or not R. ruber was added. Analysis by high-throughout sequencing showed that genera Ignavibacterium, Ferruginibacter, Limnobacter, and Denitrosoma were important during quinoline biodegradation with denitrification by QAB. In summary, bioaugmented R. ruber and endogenous bacterial strains had complementary roles when biodegrading quinoline to enhance denitrification. The significance of this study is to enable the use of industrial wastewater to provide electron donor to drive denitrification.

Anoxic/oxic treatment without biomass recycle.

The Anoxic/Oxic (A/O) process involves recirculating mixed liquor between its A and O tanks so that nitrate produced in the O tank can be used to for denitrification with influent COD in the A tank. Because biomass is recirculated along with nitrate, A/O operation leads to similar microbial communities in the A and O tanks, which may decrease the rates of denitrification and nitrification in each tank. Here, bench-scale experiments simulated this aspect of the A/O process by exchanging biomass between an anoxic flask and an oxic cylinder at exchange ratios of 0%, 20%, 30%, and 50%. Nitrification and denitrification rates were only 40% and 19% for 50% biomass exchange of that for no biomass exchange. Phylogenetic analysis documented that the microbial communities became much more similar with biomass exchange, and the finding was consistent with community composition in a full-scale A/O process in a municipal wastewater treatment plant. A two-stage vertical baffled bioreactor (VBBR) realized efficient total­nitrogen removal in recirculation without biomass exchange. Average removals of COD and TN were respectively 6% and 22% higher for the two-stage VBBR than the conventional A/O process, but its hydraulic retention time (HRT) was 55% to 70% of the volume of a conventional A/O process treating the same influent wastewater. The VBBR was more efficient because its anoxic biofilm was enriched in denitrifying bacteria, while its oxic biofilm was enriched in nitrifying bacteria. For example, the phylum Chloroflexi was greater in the An-VBBR, while the phylum Proteobacteria was greater in the Ox-VBBR.

Selective acceleration of 2-hydroxyl pyridine mono-oxygenation using specially acclimated biomass.

Biodegradation of pyridine starts with two mono-oxygenation reactions, and 2-hydroxyl pyridine (2-HP) accumulates as pyridine is mono-oxygenated in the first reaction. The accumulation of 2-HP inhibits both initial reactions. Therefore, selective acceleration of the second mono-oxygenation reaction should significantly enhance pyridine transformation and mineralization. Activated-sludge biomass was separately acclimated with pyridine or 2-HP to produce pyridine- and 2-HP-acclimated biomasses. The pyridine-acclimated biomass was superior for pyridine biodegradation, but the 2-HP-acclimated biomass was superior for 2-HP biodegradation. As a consequence, the pyridine-acclimated biomass by itself achieved faster mono-oxygenation of pyridine to 2-HP, but 2-HP accumulated, which limited mineralization to 60%. In contrast, mineralization reached 90% when one-third of the pyridine-acclimated was replaced with 2-HP-acclimated biomass, because 2-HP did not accumulate during pyridine biodegradation. The lack of 2-HP accumulation relieved its inhibition: e.g., the pyridine removal rates, normalized to the mass of pyridine-acclimated biomass, increased from 0.52 to 0.57 mM0.5⋅h-1 when one-third of the pyridine-acclimated biomass was replaced by 2-HP-acclimated biomass. Phylogenetic analysis showed that microbiological communities of pyridine-acclimated biomass and 2-HP-acclimated biomass differed in important ways. On the one hand, the 2-HP-acclimated biomass was richer and dominated by a rare biosphere, or genera having <0.1% of total reads. On the other hand, the most-enriched genus in the pyridine-acclimated community (Methylibium) is associated with the first mono-oxygenation of pyridine, while enriched genera in the 2-HP-acclimated community (Sediminibacterium and Dokdonella) are associated with the second mono-oxygenation of pyridine.

Synergy of strains that accelerate biodegradation of pyridine and quinoline.

Three bacterial strains were isolated from activated sludge acclimated to biodegrade pyridine and quinoline simultaneously. The strains were identified as Bacillus tropicus, Bacillus aquimaris, and Rhodococcus ruber. When the isolated bacteria were used for pyridine and quinoline biodegradation in separate or combined modes, R. ruber had much faster kinetics, and combining R. ruber with one or both of the Bacillus strains increased further the biodegradation kinetics. For example, the time needed for complete biodegradation of 1 mM quinoline and pyridine decreased to 20 h and 6 h, respectively, with the three strains combined, compared to 26 h and 7 h with R. ruber alone. Whereas quinoline was completely mineralized by all three strains, 10-14% of the pyridine persisted as a dead-end product, 2-hydroxypyridine (2HP). The acclimated sludge from which the three bacterial species were isolated was able to transform 2HP, and adding the bacterial strains (especially R. ruber) to the acclimated sludge accelerated the rate of 2HP removal and mineralization through a form of synergy.

How bioaugmentation with Comamonas testosteroni accelerates pyridine mono-oxygenation and mineralization.

Pyridine is a common heterocycle found in industrial wastewaters. Its biodegradation begins with a mono-oxygenation reaction, and bioaugmentation with bacteria able to carry out this mono-oxygenation is one strategy to improve pyridine removal and mineralization. Although bioaugmentation has been used to enhance the biodegradation of recalcitrant organic compounds, the specific role played by the bioaugmented bacteria usually has not been addressed. We acclimated activated-sludge biomass for pyridine biodegradation and then isolated a strain -- Comamonas testosteroni -- based on its ability to biodegrade and grow on pyridine alone. Pyridine was removed faster by C. testosteroni, compared to pyridine-acclimated biomass, but pyridine mineralization was slower. Pyridine biodegradation and mineralization rates were accelerated when C. testosteroni was bioaugmented into the acclimated biomass, which increased the amount of C. testosteroni, but otherwise had minimal effects on the microbial community. The key role of C. testosteroni was to accelerate the first step of pyridine biodegradation, mono-oxygenation to 2-hydroxylpyridine (2HP), and the acclimated biomass was better able to complete downstream reactions leading to mineralization. Thus, bioaugmentation increased the rates of pyridine mono-oxygenation and subsequent mineralization through the synergistic roles of C. testosteroni and the main community in the acclimated biomass.