Cooperation and competition between denitrification and chromate reduction in a hydrogen-based membrane biofilm reactor.

Competition and cooperation between denitrification and Cr(VI) reduction in a H2-based membrane biofilm reactor (H2-MBfR) were documented over 55 days of continuous operation. When nitrate (5 mg N/L) and chromate (0.5 mg Cr/L) were fed together, the H2-MBfR maintained approximately 100 % nitrate removal and 60 % chromate Cr(VI) removal, which means that nitrate outcompeted Cr(VI) for electrons from H2 oxidation. Removing nitrate from the influent led to an immediate increase in Cr(VI) removal (to 92 %), but Cr(VI) removal gradually deteriorated, with the removal ratio dropping to 14 % after five days. Cr(VI) removal resumed once nitrate was again added to the influent. 16S rDNA analyses showed that bacteria able to carry out H2-based denitrification and Cr(VI) reduction were in similar abundances throughout the experiment, but gene expression for Cr(VI)-reduction and export shifted. Functional genes encoding for energy-consuming chromate export (encoded by ChrA) as a means of bacterial resistance to toxicity were more abundant than genes encoding for the energy producing Cr(VI) respiration via the chromate reductase ChrR-NdFr. Thus, Cr(VI) transport and resistance to Cr(VI) toxicity depended on H2-based denitrification to supply energy. With Cr(VI) being exported from the cells, Cr(VI) reduction to Cr(III) was sustained. Thus, cooperation among H2-based denitrification, Cr(VI) export, and Cr(VI) reduction led to sustained Cr(VI) removal in the presence of nitrate, even though Cr(VI) reduction was at a competitive disadvantage for utilizing electrons from H2 oxidation.

Microalgal extracellular polymeric substances (EPS) and their roles in cultivation, biomass harvesting, and bioproducts extraction.

Microalgae extracellular polymeric substances (EPS) are complex high-molecular-weight polymers and the physicochemical properties of EPS strongly affect the core features of microalgae cultivation and resource utilization. Revealing the key roles of EPS in microalgae life-cycle processes in an interesting and novelty topic to achieve energy-efficient practical application of microalgae. This review found that EPS showed positive effect in non-gas uptake, extracellular electron transfer, toxicity resistance and heterotrophic symbiosis, but negative impact in gas transfer and light utilization during microalgae cultivation. For biomass harvesting, EPS favored biomass flocculation and large-size cell self-flocculation, but unfavored small size microalgae self-flocculation, membrane filtration, charge neutralization and biomass dewatering. During bioproducts extraction, EPS exhibited positive impact in extractant uptake, but the opposite effect in cellular membrane permeability and cell rupture. Future research on microalgal EPS were also identified, which offer suggestions for comprehensive understanding of microalgal EPS roles in various scenarios.

Achieving Long-Term Stability of Partial Nitrification and Autotrophic Denitrification in an MABR via Sulfide Dosing.

While partial nitrification (PN) has the potential to reduce energy for aeration, it has proven to be unstable when treating low-strength wastewater. This study introduces an innovative combined strategy incorporating a low rate of oxygen supply, pH control, and sulfide addition to selectively inhibit nitrite-oxidizing bacteria (NOB). This strategy led to a stable PN in a laboratory-scale membrane aerated biofilm reactor (MABR). Over a period of 260 days, the nitrite accumulation ratio exceeded 60% when treating synthetic sewage containing 50 mg NH4+-N/L. Through in situ activity testing and high-throughput sequencing, the combined strategy led to low levels of nitrite-oxidation activity (<5.5 mg N/m2 h), Nitrospira species (relative abundance <1%), and transcription of nitrite-oxidation genes (undetectable). The addition of sulfide led to simultaneous PN and autotrophic denitrification in the single-stage MABR, resulting in over 60% total inorganic nitrogen removal. Sulfur-based autotrophic denitrification consumed nitrite and inhibited NOB conversion of nitrite to nitrate. The combined strategy has potential to be applied in large-scale sewage treatment and deserves further exploration.

Biogenic Palladium Improved Perchlorate Reduction during Nitrate Co-Reduction by Diverting Electron Flow in a Hydrogenotrophic Biofilm.

Microbial reduction of perchlorate (ClO4-) is emerging as a cost-effective strategy for groundwater remediation. However, the effectiveness of perchlorate reduction can be suppressed by the common co-contamination of nitrate (NO3-). We propose a means to overcome the limitation of ClO4- reduction: depositing palladium nanoparticles (Pd0NPs) within the matrix of a hydrogenotrophic biofilm. Two H2-based membrane biofilm reactors (MBfRs) were operated in parallel in long-term continuous and batch modes: one system had only a biofilm (bio-MBfR), while the other incorporated biogenic Pd0NPs in the biofilm matrix (bioPd-MBfR). For long-term co-reduction, bioPd-MBfR had a distinct advantage of oxyanion reduction fluxes, and it particularly alleviated the competitive advantage of NO3- reduction over ClO4- reduction. Batch tests also demonstrated that bioPd-MBfR gave more rapid reduction rates for ClO4- and ClO3- compared to those of bio-MBfR. Both biofilm communities were dominated by bacteria known to be perchlorate and nitrate reducers. Functional-gene abundances reflecting the intracellular electron flow from H2 to NADH to the reductases were supplanted by extracellular electron flow with the addition of Pd0NPs.

Optimized bimetallic ratios for durable membrane catalyst-film reactors in treating nitrate-polluted water.

Nitrate contamination of surface and ground water is a significant global challenge. Most current treatment technologies separate nitrate from water, resulting in concentrated wastestreams that need to be managed. Membrane Catalyst-film Reactors (MCfR), which utilize in-situ produced nanocatalysts attached to hydrogen-gas-permeable hollow-fiber membranes, offer a promising alternative for denitrification without generating a concentrated wastestream. In hydrogen-based MCfRs, bimetallic nano-scale catalysts reduce nitrate to nitrite and then further to di-nitrogen or ammonium. This study first investigated how different molar ratios of indium-to-palladium (In:Pd) catalytic films influenced denitrification rates in batch-mode MCfRs. We evaluated eleven In-Pd bimetallic catalyst films, with In:Pd molar ratios from 0.0029 to 0.28. Nitrate-removal exhibited a volcano-shaped dependence on In content, with the highest nitrate removal (0.19 mgNO3--N-min-1 L-1) occurring at 0.045 mol In/mol Pd. Using MCfRs with the optimal In:Pd loading, we treated nitrate-spiked tap water in continuous-flow for >60 days. Nitrate removal and reduction occurred in three stages: substantial denitrification in the first stage, a decline in denitrification efficiency in the second stage, and stabilized denitrification in the third stage. Factors contributing to the slowdown of denitrification were: loss of Pd and In catalysts from the membrane surface and elevated pH due to hydroxide ion production. Sustained nitrate removal will require that these factors be mitigated.

An integrated evaluation of bioenhanced in situ LNAPL dissolution.

Performance evaluation of in situ bioremediation processes in the field is difficult due to uncertainty created by matrix and contaminant heterogeneity, inaccessibility to direct observation, expense of sampling, and limitations of some measurements. The goal of this research was to develop a strategy for evaluating in situ bioremediation of light nonaqueous-phase liquid (LNAPL) contamination and demonstrating the occurrence of bioenhanced LNAPL dissolution by: (1) integrating a suite of analyses into a rational evaluation strategy; and (2) demonstrating the strategy's application in intermediate-scale flow-cell (ISFC) experiments simulating an aquifer contaminated with a pool of LNAPL (naphthalene dissolved in dodecane). Two ISFCs were operated to evaluate how the monitored parameters changed between a "no bioremediation" scenario and an "intrinsic in situ bioremediation" scenario. Key was incorporating different measures of microbial activity and contaminant degradation relevant to bioremediation: contaminant loss; consumption of electron acceptors; and changes in total alkalinity, pH, dissolved total inorganic carbon, carbon-stable isotopes, microorganisms, and intermediate metabolites. These measurements were integrated via mass-flux modeling and mass-balance analyses to document that in situ biodegradation of naphthalene was strongly accelerated in the "intrinsic in situ bioremediation" scenario versus "no bioremediation." Furthermore, the integrated strategy provided consistent evidence of bioenhancement of LNAPL dissolution through intrinsic bioremediation by a factor of approximately 2 due to the biodegradation of the naphthalene near the pool/water interface.

Co-Removal of Perfluorooctanoic Acid and Nitrate from Water by Coupling Pd Catalysis with Enzymatic Biotransformation.

PFAS (poly- and per-fluorinated alkyl substances) represent a large family of recalcitrant organic compounds that are widely used and pose serious threats to human and ecosystem health. Here, palladium (Pd0)-catalyzed defluorination and microbiological mineralization were combined in a denitrifying H2-based membrane biofilm reactor to remove co-occurring perfluorooctanoic acid (PFOA) and nitrate. The combined process, i.e., Pd-biofilm, enabled continuous removal of ∼4 mmol/L nitrate and ∼1 mg/L PFOA, with 81% defluorination of PFOA. Metagenome analysis identified bacteria likely responsible for biodegradation of partially defluorinated PFOA: Dechloromonas sp. CZR5, Kaistella koreensis, Ochrobacterum anthropic, and Azospira sp. I13. High-performance liquid chromatography-quadrupole time-of-flight mass spectrometry and metagenome analyses revealed that the presence of nitrate promoted microbiological oxidation of partially defluorinated PFOA. Taken together, the results point to PFOA-oxidation pathways that began with PFOA adsorption to Pd0, which enabled catalytic generation of partially or fully defluorinated fatty acids and stepwise oxidation and defluorination by the bacteria. This study documents how combining catalysis and microbiological transformation enables the simultaneous removal of PFOA and nitrate.

Heterotrophic bacteria isolated from a chloraminated system accelerate chloramine decay.

This work comprehensively demonstrates the ability of heterotrophic bacteria, isolated from a chloraminated system, to decay chloramine. This study non-selectively isolated 62 cultures of heterotrophic bacteria from a water sample (0.002 mg-N/L nitrite and 1.42 mg/L total chlorine) collected from a laboratory-scale reactor system; most of the isolates (93.3%) were Mycobacterium sp. Three species of Mycobacterium and one species of Micrococcus were inoculated to a basal inorganic medium with initial concentrations of acetate (from 0 to 24 mg-C/L) and 1.5 mg/L chloramine. Bacterial growth coincided with declines in the concentrations of chloramine, acetate, and ammonium. Detailed experiments with one of the Mycobacterium sp. isolates suggest that the common mechanism of chloramine loss is auto-decomposition likely mediated by chloramine-decaying proteins. The ability of the isolates to grow and decay chloramine underscores the important role of heterotrophic bacteria in the stability of chloramine in water-distribution systems. Existing strategies based on controlling nitrification should be augmented to include minimizing heterotrophic bacteria.

Differentiation and quantification of extracellular polymeric substances from microalgae and bacteria in the mixed culture.

Extracellular polymeric substances (EPS) play significant roles in the formation, function, and interactions of microalgal-bacteria consortia. Understanding the key roles of EPS depends on reliable extraction and quantification methods, but differentiating of EPS from microalgae versus bacteria is challenging. In this work, cation exchange resin (CER) and thermal treatments were applied for total EPS extraction from microalgal-bacteria mixed culture (MBMC), flow cytometry combined with SYTOX Green staining was applied to evaluate cell disruption during EPS extraction, and auto-fluorescence-based cell sorting (AFCS) was used to separate microalgae and bacteria in the MBMC. Thermal extraction achieved much higher EPS yield than CER, but higher temperature and longer time reduced cell activity and disrupted the cells. The highest EPS yield with minimal loss of cell activity and cell disruption was achieved using thermal extraction at 55℃ for 30 min, and this protocol gave good results for MBMC with different microalgae:bacteria (M:B) mass ratios. AFCS combined with thermal treatment achieved the most-efficient biomass differentiation and low EPS loss (<4.5 %) for the entire range of M:B ratios. EPS concentrations in bacteria were larger than in microalgae: 42.8 ± 0.4 mg COD/g TSS versus 9.19 ± 0.38 mg COD/g TSS. These findings document sensitive and accurate methods to extract and quantify EPS from microalgal-bacteria aggregates.

Elemental sulfur biorecovery from phosphogypsum using oxygen-membrane biofilm reactor: Bioreactor parameters optimization, metagenomic analysis and metabolic prediction of the biofilm activity.

This work investigated elemental sulfur (S0) biorecovery from Phosphogypsum (PG) using sulfur-oxidizing bacteria in an O2-based membrane biofilm reactor (MBfR). The system was first optimized using synthetic sulfide medium (SSM) as influent, then switched to biogenic sulfide medium (BSM) generated by biological reduction of PG alkaline leachate. The results using SSM had high sulfide-oxidation efficiency (98 %), sulfide to S0 conversion (∼90 %), and S0 production rate up to 2.7 g S0/(m2.d), when the O2/S ratio was ∼0.5 g O2/g S. With the BSM influent, the system maintained high sulfide-to-S0 conversion rate (97 %), and S0-production rate of 1.6 g S0/(m2.d). Metagenomic analysis revealed that Thauera was the dominant genus in SSM and BSM biofilms. Furthermore, influent composition affected the bacterial community structure and abundances of functional microbial sulfur genes, modifying the sulfur-transformation pathways in the biofilms. Overall, this work shows promise for O2-MBfR usage in S0 biorecovery from PG-leachate and other sulfidogenic effluents.

Deconjugation potentials of natural estrogen conjugates in sewage and wastewater treatment plant: New insights from model prediction and on-site investigations.

Natural estrogen conjugates play important roles in municipal wastewater treatment plant (WWTP), but their deconjugation potentials are poorly understood. This work is the first to investigate the relationships between the enzyme activities of arylsulfatase/ß-glucuronidase and deconjugation potentials of natural estrogen conjugates. This work led to three important findings. First, the enzyme activity of ß-glucuronidase in sewage is far higher than that of arylsulfatase, while their corresponding activities in activated sludge were similar. Second, a model based on ß-glucuronidase could successfully predict the deconjugation potentials of natural estrogen glucuronide conjugates in sewage. Third, the enzyme activity of arylsulfatase in sewage was too low to lead to evident deconjugation of sulfate conjugates, which means that the deconjugation rate of estrogen sulfates can be regarded as zero. By comparing their theoretical removal based on enzyme activity and on-site investigation, it is reasonable to conclude that reverse deconjugation of estrogen conjugates (i.e., conjugation of natural estrogens to form conjugated estrogens) likely exist in WWTP, which explains well why natural estrogen conjugates cannot be effectively removed in WWTP. Meanwhile, this work provides new insights how to improve the removal performance of WWTP on natural estrogen conjugates. SYNOPSIS: This work is the first to show how arylsulfatase/ß-glucuronidase could affect deconjugation of natural estrogen conjugates and possible way to enhance their removal in wastewater treatment plant.

Method of H2 Transfer Is Vital for Catalytic Hydrodefluorination of Perfluorooctanoic Acid (PFOA).

The efficient transfer of H2 plays a critical role in catalytic hydrogenation, particularly for the removal of recalcitrant contaminants from water. One of the most persistent contaminants, perfluorooctanoic acid (PFOA), was used to investigate how the method of H2 transfer affected the catalytic hydrodefluorination ability of elemental palladium nanoparticles (Pd0NPs). Pd0NPs were synthesized through an in situ autocatalytic reduction of Pd2+ driven by H2 from the membrane. The Pd0 nanoparticles were directly deposited onto the membrane fibers to form the catalyst film. Direct delivery of H2 to Pd0NPs through the walls of nonporous gas transfer membranes enhanced the hydrodefluorination of PFOA, compared to delivering H2 through the headspace. A higher H2 lumen pressure (20 vs 5 psig) also significantly increased the defluorination rate, although 5 psig H2 flux was sufficient for full reductive defluorination of PFOA. Calculations made using density functional theory (DFT) suggest that subsurface hydrogen delivered directly from the membrane increases and accelerates hydrodefluorination by creating a higher coverage of reactive hydrogen species on the Pd0NP catalyst compared to H2 delivery through the headspace. This study documents the crucial role of the H2 transfer method in the catalytic hydrogenation of PFOA and provides mechanistic insights into how membrane delivery accelerates hydrodefluorination.

Reduction and precipitation of chromium(VI) using a palladized membrane biofilm reactor.

H2-driven reduction of hexavalent chromium (Cr(VI)) using precious-metal catalysts is promising, but its implementation in water treatment has been restricted by poor H2-transfer efficiency and high catalyst loss. We investigated the reduction of Cr(VI) through hydrogenation catalyzed by elemental-palladium nanoparticles (PdNPs) generated in-situ within biofilm of a membrane biofilm reactor (MBfR), creating a Pd-MBfR. Experiments were conducted using a Pd-MBfR and a non-Pd MBfR. The Pd-MBfR achieved Cr(VI) (1000 µg L-1) reduction of >99 % and reduced the concentration of total Cr to below 50 µg L-1, much lower than the total Cr concentration in the non-Pd MBfR effluent (290 µg L-1). The Pd-MBfR also had a lower concentration of dissolved organic compounds compared to the non-Pd MBfR, which minimized the formation of soluble organo-Cr(III) complexes and promoted precipitation of Cr(OH)3. Solid-state characterizations documented deposition of Cr(OH)3 as the product of Cr(VI) reduction in the Pd-MBfR. Metagenomic analyses revealed that the addition and reduction of Cr(VI) had minimal impact on the microbial community (dominated by Dechloromonas) and functional genes in the biofilm of the Pd-MBfR, since the PdNP-catalyzed reduction process was rapid. This study documented efficient Cr(VI) reduction and precipitation of Cr(OH)3 by the Pd-MBfR technology.

Core taxa, co-occurrence pattern, diversity, and metabolic pathways contributing to robust anaerobic biodegradation of chlorophenol.

It is hard to achieve robustness in anaerobic biodegradation of trichlorophenol (TCP). We hypothesized that specific combinations of environmental factors determine phylogenetic diversity and play important roles in the decomposition and stability of TCP-biodegrading bacteria. The anaerobic bioreactor was operated at 35 °C (H condition) or 30 °C (L condition) and mainly fed with TCP (from 28 µM to 180 µM) and organic material. Metagenome sequencing was combined with 16S rRNA gene amplicon sequencing for the microbial community analysis. The results exhibited that the property of robustness occurred in specific conditions. The corresponding co-occurrence and diversity patterns suggest high collectivization, degree and evenness for robust communities. Two types of core functional taxa were recognized: dechlorinators (unclassified Anaerolineae, Thermanaerothrix and Desulfovibrio) and ring-opening members (unclassified Proteobacteria, Methanosarcina, Methanoperedens, and Rubrobacter). The deterministic process of the expansion of niche of syntrophic bacteria at higher temperatures was confirmed. The reductive and hydrolytic dechlorination mechanisms jointly lead to C-Cl bond cleavage. H ultimately adapted to the stress of high TCP loading, with more abundant ring-opening enzyme (EC 3.1.1.45, ∼55%) and hydrolytic dechlorinase (EC 3.8.1.5, 26.5%) genes than L (∼47%, 10.5%). The functional structure (based on KEGG) in H was highly stable despite the high loading of TCP (up to 60 µM), but not in L. Furthermore, an unknown taxon with multiple functions (dechlorinating and ring-opening) was found based on genetic sequencing; its functional contribution of EC 3.8.1.5 in H (26.5%) was higher than that in L (10.5%), and it possessed a new metabolic pathway for biodegradation of halogenated aromatic compounds. This new finding is supplementary to the robust mechanisms underlying organic chlorine biodegradation, which can be used to support the engineering, regulation, and design of synthetic microbiomes.

'Dark' CO2 fixation in succinate fermentations enabled by direct CO2 delivery via hollow fiber membrane carbonation.

Anaerobic succinate fermentations can achieve high-titer, high-yield performance while fixing CO2 through the reductive branch of the tricarboxylic acid cycle. To provide the needed CO2, conventional media is supplemented with significant (up to 60 g/L) bicarbonate (HCO3-), and/or carbonate (CO32-) salts. However, producing these salts from CO2 and natural ores is thermodynamically unfavorable and, thus, energetically costly, which reduces the overall sustainability of the process. Here, a series of composite hollow fiber membranes (HFMs) were first fabricated, after which comprehensive CO2 mass transfer measurements were performed under cell-free conditions using a novel, constant-pH method. Lumen pressure and total HFM surface area were found to be linearly correlated with the flux and volumetric rate of CO2 delivery, respectively. Novel HFM bioreactors were then constructed and used to comprehensively investigate the effects of modulating the CO2 delivery rate on succinate fermentations by engineered Escherichia coli. Through appropriate tuning of the design and operating conditions, it was ultimately possible to produce up to 64.5 g/L succinate at a glucose yield of 0.68 g/g; performance approaching that of control fermentations with directly added HCO3-/CO32- salts and on par with prior studies. HFMs were further found to demonstrate a high potential for repeated reuse. Overall, HFM-based CO2 delivery represents a viable alternative to the addition of HCO3-/CO32- salts to succinate fermentations, and likely other 'dark' CO2-fixing fermentations.

A two-stage design enhanced biodegradation of high concentrations of a C16-alkyl quaternary ammonium compound in oxygen-based membrane biofilm reactors.

Quaternary ammonia compounds (QAC), such as hexadecyltrimethyl-ammonium (CTAB), are widely used as disinfectants and in personal-care products. Their use as disinfectants grew during the SARS-CoV-2 (COVID-19) pandemic, leading to increased loads to wastewater treatment systems and the environment. Though low concentrations of CTAB are biodegradable, high concentrations are toxic to bacteria. Sufficient O2 delivery is a key to achieve high CTAB removal, and the O2-based Membrane Biofilm Reactor (O2-MBfR) is a proven means to biodegrade CTAB in a bubble-free, non-foaming manner. A strategy for achieving complete biodegradation of high-concentrations of CTAB is a two-stage O2-MBfR, in which partial CTAB removal in the Lead reactor relieves inhibition in the Lag reactor. Here, more than 98 % removal of 728 mg/L CTAB could be achieved in the two-stage MBfR, and the CTAB-removal rate was 70 % higher than for a one-stage MBfR with the same O2-delivery capacity. CTAB exposure shifted the bacterial community toward Pseudomonas and Stenotrophomonas as the dominant genera. In particular, P. alcaligenes and P. aeruginosa were enriched in the Lag reactor, as they were capable of biodegrading the metabolites of initial CTAB monooxygenation. Metagenomic analysis also revealed that the Lag reactor was enriched in genes for CTAB and metabolite oxygenation, due to reduced CTAB inhibition.

A steady-state pH-control model for the biological production of elemental sulfur from sulfate in mining-influenced water.

We developed a model to predict pH, alkalinity, and the Langelier Saturation Index (LSI) in coupled systems of hydrogen-based autotrophic sulfate reduction and aerobic oxidation of sulfide to elemental sulfur. To neutralize the biologically generated base, the model allows for the addition of CO2 as part of the gas mixture, the independent addition of HCl or CO2, or a combination of the alternatives. The model was evaluated against the results from a laboratory system for the production of elemental sulfur from sulfate present in mine-tailings water, which is characterized by the presence of elevated sulfate and calcium concentrations. Model results were consistent with measurements of pH, alkalinity, and LSI. The model showed how the acid demands of the coupled reactors vary with pH, being approximately equivalent at pH over 8, when ionized sulfide predominates. Also, while the sulfidogenic reactor was well buffered due to the production of ionized sulfide, the sulfidotrophic reactor in the absence of sulfide and carbonate alkalinity was prone to pH declines. Considering that both reactors operated in the positive range of LSI, the model also indicated that addition of CO2 should be minimized due to increase in the bicarbonate concentration and its effect on increasing the LSI. Furthermore, the model also showed that exclusive reliance on HCl for pH control can be incompatible with Cl- effluent standards, which means that a compromise must be reached between CO2 and HCl additions.

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.