A novel mechanistic modelling approach for microbial selection dynamics: Towards improved design and control of raceway reactors for purple bacteria.

Purple phototrophic bacteria (PPB) show an underexplored potential for resource recovery from wastewater. Raceway reactors offer a more affordable full-scale solution on wastewater and enable useful additional aerobic processes. Current mathematical models of PPB systems provide useful mechanistic insights, but do not represent the full metabolic versatility of PPB and thus require further advancement to simulate the process for technology development and control. In this study, a new modelling approach for PPB that integrates the photoheterotrophic, and both anaerobic and aerobic chemoheterotrophic metabolic pathways through an empirical parallel metabolic growth constant was proposed. It aimed the modelling of microbial selection dynamics in competition with aerobic and anaerobic microbial community under different operational scenarios. A sensitivity analysis was carried out to identify the most influential parameters within the model and calibrate them based on experimental data. Process perturbation scenarios were simulated, which showed a good performance of the model.

Achieving Discharge Limits in Single-Stage Domestic Wastewater Treatment by Combining Urban Waste Sources and Phototrophic Mixed Cultures.

This work shows the potential of a new way of co-treatment of domestic wastewater (DWW) and a liquid stream coming from the thermal hydrolysis of the organic fraction of municipal solid waste (OFMSW) mediated by a mixed culture of purple phototrophic bacteria (PPB) capable of assimilating carbon and nutrients from the medium. The biological system is an open single-step process operated under microaerophilic conditions at an oxidative reduction potential (ORP) < 0 mV with a photoperiod of 12/24 h and fed during the light stage only so the results can be extrapolated to outdoor open pond operations by monitoring the ORP. The effluent mostly complies with the discharge values of the Spanish legislation in COD and p-values (<125 mg/L; <2 mg/L), respectively, and punctually on values in N (<15 mg/L). Applying an HRT of 3 d and a ratio of 100:7 (COD:N), the presence of PPB in the mixed culture surpassed 50% of 16S rRNA gene copies, removing 78% of COD, 53% of N, and 66% of P. Furthermore, by increasing the HRT to 5 d, removal efficiencies of 83% of COD, 65% of N, and 91% of P were achieved. In addition, the reactors were further operated in a membrane bioreactor, thus separating the HRT from the SRT to increase the specific loading rate. Very satisfactory removal efficiencies were achieved by applying an HRT and SRT of 2.3 and 3 d, respectively: 84% of COD, 49% of N, and 93% of P despite the low presence of PPB due to more oxidative conditions, which step-by-step re-colonized the mixed culture until reaching >20% of 16S rRNA gene copies after 49 d of operation. These results open the door to scaling up the process in open photobioreactors capable of treating urban wastewater and municipal solid waste in a single stage and under microaerophilic conditions by controlling the ORP of the system.

Assessment of Voltage Influence in Carbon Dioxide Fixation Process by a Photo-Bioelectrochemical System under Photoheterotrophy.

Bioelectrochemical systems are a promising technology capable of reducing CO2 emissions, a renewable carbon source, using electroactive microorganisms for this purpose. Purple Phototrophic Bacteria (PPB) use their versatile metabolism to uptake external electrons from an electrode to fix CO2. In this work, the effect of the voltage (from -0.2 to -0.8 V vs. Ag/AgCl) on the metabolic CO2 fixation of a mixed culture of PPB under photoheterotrophic conditions during the oxidation of a biodegradable carbon source is demonstrated. The minimum voltage to fix CO2 was between -0.2 and -0.4 V. The Calvin-Benson-Bassham (CBB) cycle is the main electron sink at these voltages. However, lower voltages caused the decrease in the current intensity, reaching a minimum at -0.8 V (-4.75 mA). There was also a significant relationship between the soluble carbon uptake in terms of chemical oxygen demand and the electron consumption for the experiments performed at -0.6 and -0.8 V. These results indicate that the CBB cycle is not the only electron sink and some photoheterotrophic metabolic pathways are also being affected under electrochemical conditions. This behavior has not been tested before in photoheterotrophic conditions and paves the way for the future development of photobioelectrochemical systems under heterotrophic conditions.

Substrate availability drives mixed culture fermentation of glucose to lactate at steady state.

Mixed-culture fermentation (MCF) enables carbon recycling from complex organic waste streams into valuable feedstock chemicals. Using complex microbial consortia, MCF systems can be tuned to produce a range of biochemicals to meet market demand. However, the metabolic mechanisms and community interactions which drive biochemical production changes under differing conditions are currently poorly understood. These mechanisms are critical to useful MCF production models. Furthermore, predictable product transitions are currently limited to pH-driven changes between butyrate and ethanol, and chain-elongation (fed by lactate, acetate, and ethanol) to butyrate, valerate, and hexanoate. Lactate, a high-value biopolymer feedstock chemical, has been observed in transition states, but sustained production has not been described. In this study, steady state lactate production was achieved by increasing the organic loading rate of a butyrate-producing system from limiting to nonlimiting conditions at pH 5.5. Crucially, butyrate production resumed upon return to substrate-limited conditions. 16S ribosomal DNA community profiling combined with metaproteomics demonstrated that the butyrate-producing lineage Megasphaera redirected carbon flow through the methylglyoxal bypass when substrate was nonlimiting, which altered the community structure and metabolic expression toward lactate production. This metabolic mechanism can be included in future MCF models to describe the changes in product generation in substrate nonlimiting conditions.

Purple phototrophic bacteria for resource recovery: Challenges and opportunities.

Sustainable development is driving a rapid focus shift in the wastewater and organic waste treatment sectors, from a "removal and disposal" approach towards the recovery and reuse of water, energy and materials (e.g. carbon or nutrients). Purple phototrophic bacteria (PPB) are receiving increasing attention due to their capability of growing photoheterotrophically under anaerobic conditions. Using light as energy source, PPB can simultaneously assimilate carbon and nutrients at high efficiencies (with biomass yields close to unity (1 g CODbiomass·g CODremoved-1)), facilitating the maximum recovery of these resources as different value-added products. The effective use of infrared light enables selective PPB enrichment in non-sterile conditions, without competition with other phototrophs such as microalgae if ultraviolet-visible wavelengths are filtered. This review reunites results systematically gathered from over 177 scientific articles, aiming at producing generalized conclusions. The most critical aspects of PPB-based production and valorisation processes are addressed, including: (i) the identification of the main challenges and potentials of different growth strategies, (ii) a critical analysis of the production of value-added compounds, (iii) a comparison of the different value-added products, (iv) insights into the general challenges and opportunities and (v) recommendations for future research and development towards practical implementation. To date, most of the work has not been executed under real-life conditions, relevant for full-scale application. With the savings in wastewater discharge due to removal of organics, nitrogen and phosphorus as an important economic driver, priorities must go to using PPB-enriched cultures and real waste matrices. The costs associated with artificial illumination, followed by centrifugal harvesting/dewatering and drying, are estimated to be 1.9, 0.3-2.2 and 0.1-0.3 $·kgdry biomass-1. At present, these costs are likely to exceed revenues. Future research efforts must be carried out outdoors, using sunlight as energy source. The growth of bulk biomass on relatively clean wastewater streams (e.g. from food processing) and its utilization as a protein-rich feed (e.g. to replace fishmeal, 1.5-2.0 $·kg-1) appears as a promising valorisation route.

A systematic optimization of piggery wastewater treatment with purple phototrophic bacteria.

The increase in natural water bodies pollution caused by intensive animal farming requires the development of innovative sustainable treatment processes. This study assessed the influence of piggery wastewater (PWW) load, air dosing, CO2/NaHCO3- supplementation and pH control on PWW treatment by mixed cultures of purple phototrophic bacteria (PPB) under infrared radiation in batch photobioreactors. PPB was not able to grow in raw PWW but PWW dilution prevented inhibition and supported an effective light penetration. Despite the fact that PPB were tolerant to O2, carbon recovery decreased in the presence of air (induced by stripping). CO2 supplementation was identified as an effective strategy to maximize the removal of carbon during PPB-based PWW treatment with removal efficiencies of 72% and 74% for TOC and VFAs. However, the benefits derived from CO2 addition were induced by the indirect pH control exerted in the cultivation medium. Thus, PPB supported an optimal pollutant removal performance at pH 7, with removal efficiencies of 75%, 39% and 98% for TOC, TN and VFAs.

Anaerobic digestion of purple phototrophic bacteria - The release step of the partition-release-recover concept.

Purple phototrophic bacteria (PPB) have been proposed as a high-growth, assimilative option for wastewater treatment. The original partition-release-recover concept proposal requires their near complete digestion and release (and subsequent recovery) of energy and nutrients in an anaerobic digester. While the growth (partition) step has been extensively assessed, no work has been done on their anaerobic digestion characteristics (release). Continuous mesophilic (20d) and thermophilic (10d) digestion could achieve around 55% volatile solids degradation (VSD), with 35% (mesophilic) and 20% (thermophilic) nitrogen solubilisation. Post digestion (with/without pretreatment) could increase the VSD to 70% and nitrogen solubilisation to 43%. A number of pretreatment options were tested, with high temperature and sonication being relatively effective, and chemical treatment, and temperature phased digestion being relatively ineffective vs controls. Overall, anaerobic digestion of PPB results in substantial residual particulate material, with an increased nitrogen content, and avenues to effectively utilise this residue should be identified.

Novel approach for the treatment of the organic fraction of municipal solid waste: Coupling thermal hydrolysis with anaerobic digestion and photo-fermentation.

In this study, two different organic fractions of municipal solid waste (OFMSW) served as raw material in a novel treatment process that combines thermal hydrolysis (TH) pretreatment at different times, followed by anaerobic digestion of the solid fraction and photo-fermentation of the liquid fraction. The results indicate that both wastes performed similarly, and no statistically relevant differences stand out on the overall performance regarding TH times. The thermal pretreatment improves the biodegradability of the solid fraction during anaerobic digestion compensating the loss of the organic matter in the liquid fraction. The produced biogas may feed a combined heat and power (CHP) system, making the process energetically positive in all studied scenarios. In addition, the combination of TH and anaerobic digestion decreased the volume of the waste to be disposed by 59-61%, which is 5-11% higher than that obtained with the traditional treatment of anaerobic digestion process. Specific phototrophic activity tests were performed on the liquid phase using a mixed culture of purple phototrophic bacteria (PPB) that consumed up to 80% of the soluble organics. The assays yielded an average 52% efficiency on specific phototrophic activity (kM) and 62% on biomass yield (YX/S), compared to an optimized growth medium. PPB was also capable of producing polyhydroxyalkanoates, bioH2 and single-cell protein without optimization. Apart from methane, the overall mass balances showed yields up to 150 g of high added-value products per Kg of initial total solids on this proof-of-concept platform.

Assessing the potential of purple phototrophic bacteria for the simultaneous treatment of piggery wastewater and upgrading of biogas.

The potential of purple phototrophic bacteria (PPB) for the simultaneous treatment of piggery wastewater (PWW) and biogas upgrading was evaluated batchwise in gas-tight photobioreactors. PWW dilution was identified as a key parameter determining the efficiency of wastewater treatment and biomethane quality in PPB photobioreactors. Four times diluted PWW supported the most efficient total organic carbon (TOC) and total nitrogen removals (78% and 13%, respectively), with CH4 concentrations of 90.8%. The influence of phosphorous concentration (supplementation of 50 mg L-1 of P-PO43-) on PPB-based PWW treatment coupled to biogas upgrading was investigated. TOC removals of ≈60% and CH4 concentrations of ≈90.0% were obtained regardless of phosphorus supplementation. Finally, the use of PPB and algal-bacterial consortia supported CH4 concentrations in the upgraded biogas of 93.3% and 73.6%, respectively, which confirmed the potential PPB for biogas upgrading coupled to PWW treatment.

Resource Recovery Potential From Lignocellulosic Feedstock Upon Lysis With Ionic Liquids.

Lignocellulosic residues from energy crops offer a high potential to recover bioproducts and biofuels that can be used as raw matter for agriculture activities within a circular economy framework. Anaerobic digestion (AD) is a well-established driver to convert these residues into energy and bioproducts. However, AD of lignocellulosic matter is slow and yields low methane potential, and therefore several pre-treatment methods have been proposed to increase the energy yield of this process. Hereby, we have assessed the pre-treatment of lignocellulosic biomass (barley straw) with the ionic liquid (IL) 1-ethyl-3-methylimidazolium acetate and its effect on the biochemical methane potential (BMP). The BMP of the residue was evaluated at different inoculum to substrate (I/S) ratios and working under meso and thermophilic conditions. Solids destruction upon AD is highly enhanced by the IL-pretreatment. This also resulted in a higher BMP, both in mesophilic as well as thermophilic conditions. At the optimum I/S ratio of 2:1 (dried weight, dw), the BMP of the IL-pre-treated feedstock increased 28 and 80% for 35 days of thermophilic and mesophilic AD, respectively, as compared to the fresh feedstock, achieving values of 364 and 412 LCH4/kgTS. We also explored the effect of this pretreatment on the phosphorus recovery potential from the digestate upon release from the AD process. Thermophilic anaerobic digestion of IL-pre-treated biomass provided the highest P recovery potential from lignocellulosic residues (close to 100% of the theoretical P content of the lignocellulosic feedstock). Therefore, the pretreatment of lignocellulosic feedstock with IL before AD is a promising platform to obtain bioenergy and recover P to be regained for the agriculture sector.

White and infrared light continuous photobioreactors for resource recovery from poultry processing wastewater - A comparison.

Concentrated wastewaters from agricultural industries represent a key opportunity for the upcycling of organics, nitrogen and phosphorus to higher value products such as microbial protein. Phototrophic or photosynthetic microbes very effectively capture input organics and nutrients as microbial protein. This study compares purple phototrophic bacteria (PPB) and microalgae (photosynthesis) for this purpose, treating real, high strength poultry processing wastewater in continuous photo bioreactors utilising infrared (IR) and white light (WL) respectively. Both reactors could effectively treat the wastewaters, and at similar loading rates (4 kgCOD m-3d-1). The infrared reactor (IRR) was irradiated at 18 W m-2 and the white light reactor (WLR) reactor at 1.5-2 times this. The IRR could remove up to 90% total chemical oxygen demand (TCOD), 90% total nitrogen (TN) and 45% total phosphorus (TP) at 1.0 d hydraulic retention time (HRT) and recover around 190 kg of crude protein per tonne of influent COD at 7.0 kWh per dry tonne-1 light input, with PPB dominating all samples. In comparison, the WLR removed up to 98% COD, 94% TN and 44% TP at 43-90% higher irradiance compared to the PPB reactor. Microalgae did not dominate the WLR and the community was instead a mix of microbes (algae, bacteria, zooplankton and detritus - ALBAZOD) with a production of approximately 140 kg crude protein per tonne influent COD.

Removal of pharmaceutical compounds from urban wastewater by an advanced bio-oxidation process based on fungi Trametes versicolor immobilized in a continuous RBC system.

Conventional wastewater treatment plants (WWTPs) are not able to remove completely some emerging contaminants, such as residual pharmaceutical compounds (PCs) with potential ecotoxicity to water bodies. An advanced bio-oxidation process (ABOP) using white-rot fungi (WRF) has been proposed as alternative biological treatment for degradation of non-biodegradable compounds. A synthetic and real wastewater spiked with 12 PCs at 50 µg L-1 was treated by means of ABOP based on WRF in a rotating biological contactor (RBC) at 1 day of hydraulic retention time (HRT). The ABOP achieved a remarkable biological performance in terms of TOC removal and reduction of N-NH4 + and P-PO4 3- nutrients. Likewise, 5 of the 12 PCs were eliminated with removal efficiencies ranging from 80 to 95%, whereas 6 of 12 PCs were eliminated with removal values ranging from 50 to 70%. The anaerobic digestion of the fungal sludge generated upon the treatment was also evaluated, obtaining a methane yield of 250 mL CH4 gVS -1. These results evidenced that the proposed ABOP is a promising alternative for the sustainable wastewater treatment of urban effluents, combining advanced oxidation with biological operation for the removal of emerging PCs and energy recovery.

Elemental copper nanoparticle toxicity to anaerobic ammonium oxidation and the influence of ethylene diamine-tetra acetic acid (EDTA) on copper toxicity.

Soluble ions released by elemental copper nanoparticles (Cu0 NP) are toxic to key microorganisms of wastewater treatment processes. However, their toxicity to anaerobic ammonium oxidation (anammox) has not yet been studied. Chelating agents occurring in wastewater may decrease copper ions (Cu2+) concentration and consequently, decrease copper toxicity. This study evaluated Cu0 NP and CuCl2 toxicity to anammox and the influence of ethylene diamine-tetra acetic acid (EDTA) on copper toxicity. Bioassays were supplemented with Cu0 NP or CuCl2 with and without EDTA. Anammox activities were used to calculate inhibition constants (Ki). Results showed that Cu0 NP are toxic to anammox. Ki constants with respect to added copper were 1.8- and 2.81-fold larger (less toxic) in EDTA-containing assays for Cu0 NP and CuCl2, respectively, compared to EDTA-free assays. Additionally, Ki constants calculated in EDTA-free assays with respect the measured dissolved copper concentration were 0.023 mM Cu0 NP and 0.014 mM CuCl2. The similarity of these Ki constants indicates that Cu0 NP toxicity to anammox is caused by the release of Cu2+. Finally, severe toxicity caused by 0.315 mM and Cu0 NP 0.118 mM CuCl2 was attenuated by 88-100% when 0.14 mM EDTA was supplied. Toxicity attenuation likely occurred because EDTA complexed Cu2+ ions, thus, decreasing their bioavailability. Overall, this study indicates that Cu0 NP and CuCl2 are toxic to anammox, and furthermore, that EDTA attenuates Cu0 NP and CuCl2 toxicity to anammox by complexing Cu2+ ions.

Low temperature treatment of domestic wastewater by purple phototrophic bacteria: Performance, activity, and community.

Low wastewater temperatures affect microbial growth rates and microbial populations, as well as physical chemical characteristics of the wastewater. Wastewater treatment plant design needs to accommodate changing temperatures, and somewhat limited capacity is a key criticism of low strength anaerobic treatment such as Anaerobic Membrane Bioreactors (AnMBR). This study evaluates the applicability of an alternative platform utilizing purple phototrophic bacteria for low temperature domestic wastewater treatment. Two photo-anaerobic membrane bioreactors (PAnMBR) at ambient (22 °C) and low temperatures (10 °C) were compared to fully evaluate temperature response of critical processes. The results show good functionality at 10 °C in comparison with ambient operation. This enabled operation at 10 °C to discharge limits (TCOD < 100 mg L(-1); TN < 10 mg L(-1) and TP < 1 mg L(-1)) at a HRT < 1 d. While capacity of the system was not limited, microbial community showed a strong shift to a far narrower diversity, almost complete dominance by PPB, and of a single Rhodobacter spp. compared to a more diverse community in the ambient reactor. The outcomes of the current work enable applicability of PPB for domestic wastewater treatment to a broad range of regions.

Domestic wastewater treatment with purple phototrophic bacteria using a novel continuous photo anaerobic membrane bioreactor.

A key future challenge of domestic wastewater treatment is nutrient recovery while still achieving acceptable discharge limits. Nutrient partitioning using purple phototrophic bacteria (PPB) has the potential to biologically concentrate nutrients through growth. This study evaluates the use of PPB in a continuous photo-anaerobic membrane bioreactor (PAnMBR) for simultaneous organics and nutrient removal from domestic wastewater. This process could continuously treat domestic wastewater to discharge limits (<50 mgCOD L(-1), 5 mgN L(-1), 1.0 mgP L(-1)). Approximately 6.4 ± 1.3 gNH4-N and 1.1 ± 0.2 gPO4-P for every 100 gSCOD were removed at a hydraulic retention time of 8-24 h and volumetric loading rates of 0.8-2.5 COD kg m(3) d(-1). Thus, a minimum of 200 mg L(-1) of ethanol (to provide soluble COD) was required to achieve these discharge limits. Microbial community through sequencing indicated dominance of >60% of PPB, though the PPB community was highly variable. The outcomes from the current work demonstrate the potential of PPB for continuous domestic (and possibly industrial) wastewater treatment and nutrient recovery. Technical challenges include the in situ COD supply in a continuous reactor system, as well as efficient light delivery. Addition of external (agricultural or fossil) derived organics is not financially nor environmentally justified, and carbon needs to be sourced internally from the biomass itself to enable this technology. Reduced energy consumption for lighting is technically feasible, and needs to be addressed as a key objective in scaleup.

Resource Recovery from Wastewater by Biological Technologies: Opportunities, Challenges, and Prospects.

Limits in resource availability are driving a change in current societal production systems, changing the focus from residues treatment, such as wastewater treatment, toward resource recovery. Biotechnological processes offer an economic and versatile way to concentrate and transform resources from waste/wastewater into valuable products, which is a prerequisite for the technological development of a cradle-to-cradle bio-based economy. This review identifies emerging technologies that enable resource recovery across the wastewater treatment cycle. As such, bioenergy in the form of biohydrogen (by photo and dark fermentation processes) and biogas (during anaerobic digestion processes) have been classic targets, whereby, direct transformation of lipidic biomass into biodiesel also gained attention. This concept is similar to previous biofuel concepts, but more sustainable, as third generation biofuels and other resources can be produced from waste biomass. The production of high value biopolymers (e.g., for bioplastics manufacturing) from organic acids, hydrogen, and methane is another option for carbon recovery. The recovery of carbon and nutrients can be achieved by organic fertilizer production, or single cell protein generation (depending on the source) which may be utilized as feed, feed additives, next generation fertilizers, or even as probiotics. Additionlly, chemical oxidation-reduction and bioelectrochemical systems can recover inorganics or synthesize organic products beyond the natural microbial metabolism. Anticipating the next generation of wastewater treatment plants driven by biological recovery technologies, this review is focused on the generation and re-synthesis of energetic resources and key resources to be recycled as raw materials in a cradle-to-cradle economy concept.

Elemental copper nanoparticle toxicity to different trophic groups involved in anaerobic and anoxic wastewater treatment processes.

Elemental copper nanoparticles (Cu(0) NPs) are potentially inhibitory to the different key microbial trophic groups involved in biological wastewater treatment processes. Cu-based NPs are known to be toxic to methanogens at low concentrations. However, very little is known about the toxic effect of Cu(0) NPs on other microbial groups involved in either upper trophic levels of anaerobic digestion or anoxic nitrogen removal processes. This study evaluated the toxicity of Cu(0) NPs to glucose fermentation, syntrophic propionate oxidation and denitrification in shaken batch bioassays with soluble substrates. Batch experiments were also supplemented with CuCl2 to evaluate the inhibitory impact of soluble Cu(II) ions. Syntrophic propionate oxidation and glucose fermentation were the least and most inhibited processes with inhibition constant (Ki) values of 0.202 and 0.047 mM of added Cu(0) NPs, respectively. Further analyses revealed that the Ki values calculated as a function of the free soluble Cu concentration were <0.003 mM for every biological process tested and most of these Ki values were similar in order of magnitude regardless of whether the Cu source was CuCl2 or Cu(0) NPs. The results taken as a whole indicate that Cu(0) NPs are toxic to all the microbial processes studied. Therefore, Cu(0) NPs can potentially be an important inhibitor of anaerobic wastewater treatment processes that rely on these trophic groups. The evidence suggests that the inhibitory impact of Cu(0) NPs was mainly due to the release of toxic Cu(II) ions originating from the corrosion and dissolution of Cu(0) NPs.

Role of biogenic sulfide in attenuating zinc oxide and copper nanoparticle toxicity to acetoclastic methanogenesis.

Soluble ions released by zinc oxide (ZnO) and copper (Cu(0)) nanoparticles (NPs) have been associated with toxicity to methanogens. This study evaluated the role of biogenic sulfide in attenuating ZnO and Cu(0) NP toxicity to methanogens. Short- and long-term batch experiments were conducted to explore ZnO and Cu(0) NPs toxicity to acetoclastic methanogens in sulfate-containing (0.4mM) and sulfate-free conditions. ZnO and Cu(0) were respectively 14 and 7-fold less toxic in sulfate-containing than in sulfate-free assays as indicated by inhibitory constants (Ki). The Ki with respect to residual soluble metal indicated that soluble metal was well correlated with toxicity irrespective of the metal ion source or presence of biogenic sulfide. Long-term assays indicated that ZnO and Cu(0) NPs caused different effects on methanogens. ZnO NPs without protection of sulfide caused a chronic effect, whereas Cu(0) NPs caused an acute effect and recovered. This study confirms that biogenic sulfide effectively attenuates ZnO and Cu(0) NPs toxicity to methanogens by the formation of metal sulfides.

The intracellular proton gradient enables anaerobic ammonia oxidizing (anammox) bacteria to tolerate NO2 - inhibition.

Anammox bacteria are inhibited by nitrite, which is one of their substrates. By utilizing 2,4 dinitrophenol and carbonyl cyanide m-chlorophenyl hydrazone, two uncouplers of respiration, we demonstrate that nitrite tolerance of anammox cells is strongly dependent on their ability to maintain a proton gradient, which may be the driving force for active nitrite transport system.