Oxygen-limited Autotrophic Nitrification/denitrification Research Articles

Comammox rather than AOB dominated the efficient autotrophic nitrification-denitrification process in an extremely oxygen-limited environment

The discovery of complete ammonia oxidizer (comammox) has challenged the traditional understanding of the two-step nitrification process. However, their functions in the oxygen-limited autotrophic nitrification-denitrification (OLAND) process remain unclear. In this study, OLAND was achieved using comammox-dominated nitrifying bacteria in an extremely oxygen-limited environment with a dissolved oxygen concentrations of 0.05 mg/L. The ammonia removal efficiency exceeded 97 %, and the total nitrogen removal efficiency reached 71 % when sodium bicarbonate was used as the carbon source. The pseudo-first- and second-order models were found to best fit the ammonia removal processes under low and high loads, respectively, suggesting distinct ammonia removal pathways. Full-length 16S rRNA gene sequencing and metagenomic results revealed that comammox-dominated under different oxygen levels, in conjunction with anammox and heterotrophic denitrifiers. The abundance of enzymes involved in energy metabolism indicates the coexistence of anammox and autotrophic nitrification-heterotrophic denitrification pathways. The binning results showed that comammox bacteria engaged in horizontal gene transfer with nitrifiers, anammox bacteria, and denitrifiers to adapt to an obligate environments. Therefore, this study demonstrated that comammox, anammox, and heterotrophic denitrifiers play important roles in the OLAND process and provide a reference for further reducing aeration energy in the autotrophic nitrogen removal process.

Fate of pharmaceuticals in full-scale source separated sanitation system

Removal of 14 pharmaceuticals and 3 of their transformation products was studied in a full-scale source separated sanitation system with separate collection and treatment of black water and grey water. Black water is treated in an up-flow anaerobic sludge blanket (UASB) reactor followed by oxygen-limited autotrophic nitrification–denitrification in a rotating biological contactor and struvite precipitation. Grey water is treated in an aerobic activated sludge process. Concentration of 10 pharmaceuticals and 2 transformation products in black water ranged between low μg/l to low mg/l. Additionally, 5 pharmaceuticals were also present in grey water in low μg/l range. Pharmaceutical influent loads were distributed over two streams, i.e. diclofenac was present for 70% in grey water, while the other compounds were predominantly associated to black water. Removal in the UASB reactor fed with black water exceeded 70% for 9 pharmaceuticals out of the 12 detected, with only two pharmaceuticals removed by sorption to sludge. Ibuprofen and the transformation product of naproxen, desmethylnaproxen, were removed in the rotating biological contactor. In contrast, only paracetamol removal exceeded 90% in the grey water treatment system while removal of other 7 pharmaceuticals was below 40% or even negative. The efficiency of pharmaceutical removal in the source separated sanitation system was compared with removal in the conventional sewage treatment plants. Furthermore, effluent concentrations of black water and grey water treatment systems were compared with predicted no-effect concentrations to assess toxicity of the effluent. Concentrations of diclofenac, ibuprofen and oxazepam in both effluents were higher than predicted no-effect concentrations, indicating the necessity of post-treatment. Ciprofloxacin, metoprolol and propranolol were found in UASB sludge in μg/g range, while pharmaceutical concentrations in struvite did not exceed the detection limits.

The manufacturing microbe.

In all industrialized countries, the manufacturing industry appears to be in difficulty: the costs of maintaining skilled workers to do complicated technical tasks are becoming a major hurdle. The fact that a majority of the young professionals will spend their whole lives just typing in front of a screen is worrying plenty of sociologists, particularly because of the simple fact that our brains apparently are sparked to creativity by being involved in complex handling – read manufacturing – exercises (Sennett, 2014).

Optimized cryopreservation of mixed microbial communities for conserved functionality and diversity.

The use of mixed microbial communities (microbiomes) for biotechnological applications has steadily increased over the past decades. However, these microbiomes are not readily available from public culture collections, hampering their potential for widespread use. The main reason for this lack of availability is the lack of an effective cryopreservation protocol. Due to this critical need, we evaluated the functionality as well as the community structure of three different types of microbiomes before and after cryopreservation with two cryoprotective agents (CPA). Microbiomes were selected based upon relevance towards applications: (1) a methanotrophic co-culture (MOB), with potential for mitigation of greenhouse gas emissions, environmental pollutants removal and bioplastics production; (2) an oxygen limited autotrophic nitrification/denitrification (OLAND) biofilm, with enhanced economic and ecological benefits for wastewater treatment, and (3) fecal material from a human donor, with potential applications for fecal transplants and pre/probiotics research. After three months of cryopreservation at −80°C, we found that metabolic activity, in terms of the specific activity recovery of MOB, aerobic ammonium oxidizing bacteria (AerAOB) and anaerobic AOB (AnAOB, anammox) in the OLAND mixed culture, resumes sooner when one of our selected CPA [dimethyl sulfoxide (DMSO) and DMSO plus trehalose and tryptic soy broth (DMSO+TT)] was added. However, the activity of the fecal community was not influenced by the CPA addition, although the preservation of the community structure (as determined by 16S rRNA gene sequencing) was enhanced by addition of CPA. In summary, we have evaluated a cryopreservation protocol that succeeded in preserving both community structure and functionality of value-added microbiomes. This will allow individual laboratories and culture collections to boost the use of microbiomes in biotechnological applications.

Control of nitratation in an oxygen-limited autotrophic nitrification/denitrification rotating biological contactor through disc immersion level variation

With oxygen supply playing a crucial role in an oxygen-limited autotrophic nitrification/denitrification (OLAND) rotating biological contactor (RBC), its controlling factors were investigated in this study. Disc rotation speeds (1.8 and 3.6rpm) showed no influence on the process performance of a lab-scale RBC, although abiotic experiments showed a significant effect on the oxygenation capacity. Estimations of the biological oxygen uptake rate revealed that 85-89% of the oxygen was absorbed by the microorganisms during the air exposure of the discs. Indeed, increasing the disc immersion (50 to 75-80%) could significantly suppress undesired nitratation, on the short and long term. The presented results demonstrated that nitratation could be controlled by the immersion level and revealed that oxygen control in an OLAND RBC should be predominantly based on the atmospheric exposure percentage of the discs.

One-stage partial nitritation/anammox at 15 °C on pretreated sewage: feasibility demonstration at lab-scale

Energy-positive sewage treatment can be achieved by implementation of oxygen-limited autotrophic nitrification/denitrification (OLAND) in the main water line, as the latter does not require organic carbon and therefore allows maximum energy recovery through anaerobic digestion of organics. To test the feasibility of mainstream OLAND, the effect of a gradual temperature decrease from 29 to 15 °C and a chemical oxygen demand (COD)/N increase from 0 to 2 was tested in an OLAND rotating biological contactor operating at 55-60 mg NH₄(+)-N L(-1) and a hydraulic retention time of 1 h. Moreover, the effect of the operational conditions and feeding strategies on the reactor cycle balances, including NO and N₂O emissions were studied in detail. This study showed for the first time that total nitrogen removal rates of 0.5 g N L(-1) day(-1) can be maintained when decreasing the temperature from 29 to 15 °C and when low nitrogen concentration and moderate COD levels are treated. Nitrite accumulation together with elevated NO and N₂O emissions (5% of N load) were needed to favor anammox compared with nitratation at low free ammonia (<0.25 mg N L(-1)), low free nitrous acid (<0.9 μg N L(-1)), and higher DO levels (3-4 mg O₂ L(-1)). Although the total nitrogen removal rates showed potential, the accumulation of nitrite and nitrate resulted in lower nitrogen removal efficiencies (around 40%), which should be improved in the future. Moreover, a balance should be found in the future between the increased NO and N₂O emissions and a decreased energy consumption to justify OLAND mainstream treatment.

Enrichment of anaerobic ammonium oxidizing (Anammox) bacteria from OLAND and conventional sludge: Features and limitations

The discharge of nitrogen is increasingly restricted. The autotrophic nitrogen removal process, which combines partial nitritation and anaerobic ammonium oxidation (Anammox), is recommended for high ammonium loaded streams with no or low organic carbon content because of the lower operational costs. However, the cultivation and start-up period can be long and time-consuming due to the slow growth rate of Anammox bacteria.The aim of this research was to evaluate the feasibility of Anammox cultivation in an anaerobic continuous stirred tank reactor (CSTR). Monitoring of nitrogen conversion showed Anammox activity with an increasing nitrogen removal rate up to 700mgNL−1day−1 in a reactor inoculated with OLAND (Oxygen-Limited Autotrophic Nitrification/Denitrification) seed sludge. A second lab scale reactor, a modified CSTR which was operated as an anaerobic upflow fixed bed reactor (UFBR), was inoculated with nitrifying and denitrifying sludge from a manure treatment plant. The fixed bed consisted of polyurethane sponge which served as a carrier material for the slowly growing biomass. The UFBR showed removal according to the Anammox metabolism within 3months and a nitrogen removal rate up to 250mgNL−1day−1 was achieved. Primary research on the treatment on pig manure wastewater indicates that organic load had an inhibitory effect on the Anammox activity. The results also indicate that Anammox bacteria can stay dormant or hidden for longer periods, but will reactivate under the suitable conditions.

Efficient Total Nitrogen Removal in an Ammonia Gas Biofilter through High-Rate OLAND

Ammonia gas is conventionally treated in nitrifying biofilters; however, addition of organic carbon to perform post-denitrification is required to obtain total nitrogen removal. Oxygen-limited autotrophic nitrification/denitrification (OLAND), applied in full-scale for wastewater treatment, can offer a cost-effective alternative for gas treatment. In this study, the OLAND application thus was broadened toward ammonia loaded gaseous streams. A down flow, oxygen-saturated biofilter (height of 1.5 m; diameter of 0.11 m) was fed with an ammonia gas stream (248 ± 10 ppmv) at a loading rate of 0.86 ± 0.04 kg N m(-3) biofilter d(-1) and an empty bed residence time of 14 s. After 45 days of operation a stable nitrogen removal rate of 0.67 ± 0.06 kg N m(-3) biofilter d(-1), an ammonia removal efficiency of 99%, a removal of 75-80% of the total nitrogen, and negligible NO/N(2)O productions were obtained at water flow rates of 1.3 ± 0.4 m(3) m(-2) biofilter section d(-1). Profile measurements revealed that 91% of the total nitrogen activity was taking place in the top 36% of the filter. This study demonstrated for the first time highly effective and sustainable autotrophic ammonia removal in a gas biofilter and therefore shows the appealing potential of the OLAND process to treat ammonia containing gaseous streams.

OLAND is feasible to treat sewage-like nitrogen concentrations at low hydraulic residence times

Energy-positive sewage treatment can, in principle, be obtained by maximizing energy recovery from concentrated organics and by minimizing energy consumption for concentration and residual nitrogen removal in the main stream. To test the feasibility of the latter, sewage-like nitrogen influent concentrations were treated with oxygen-limited autotrophic nitrification/denitrification (OLAND) in a lab-scale rotating biological contactor at 25°C. At influent ammonium concentrations of 66 and 29 mg N L(-1) and a volumetric loading rate of 840 mg N L(-1) day(-1) yielding hydraulic residence times (HRT) of 2.0 and 1.0 h, respectively, relatively high nitrogen removal rates of 444 and 383 mg N L(-1) day(-1) were obtained, respectively. At low nitrogen levels, adapted nitritation and anammox communities were established. The decrease in nitrogen removal was due to decreased anammox and increased nitratation, with Nitrospira representing 6% of the biofilm. The latter likely occurred given the absence of dissolved oxygen (DO) control, since decreasing the DO concentration from 1.4 to 1.2 mg O(2) L(-1) decreased nitratation by 35% and increased anammox by 32%. Provided a sufficient suppression of nitratation, this study showed the feasibility of OLAND to treat low nitrogen levels at low HRT, a prerequisite to energy-positive sewage treatment.

Long-chain acylhomoserine lactones increase the anoxic ammonium oxidation rate in an OLAND biofilm

The oxygen-limited autotrophic nitrification/denitrification (OLAND) process comprises one-stage partial nitritation and anammox, catalyzed by aerobic and anoxic ammonium-oxidizing bacteria (AerAOB and AnAOB), respectively. The goal of this study was to investigate whether quorum sensing influences anoxic ammonium oxidation in an OLAND biofilm, with AnAOB colonizing 13% of the biofilm, as determined with fluorescent in situ hybridization (FISH). At high biomass concentrations, the specific anoxic ammonium oxidation rate of the OLAND biofilm significantly increased with a factor of 1.5 ± 0.2 compared to low biomass concentrations. Supernatant obtained from the biofilm showed no ammonium-oxidizing activity on itself, but its addition to low OLAND biomass concentrations resulted in a significant activity increase of the biomass. In the biofilm supernatant, the presence of long-chain acylhomoserine lactones (AHLs) was shown using the reporter strain Chromobacterium violaceum CV026, and one specific AHL, N-dodecanoyl homoserine lactone (C(12)-HSL), was identified via LC-MS/MS. Furthermore, C(12)-HSL was detected in an AnAOB-enriched community, but not in an AerAOB-enriched community. Addition of C(12)-HSL to low OLAND biomass concentrations resulted in a significantly higher ammonium oxidation rate (p < 0.05). To our knowledge, this is the first report demonstrating that AHLs enhance the anoxic ammonium oxidation process. Future work should confirm which species are responsible for the in situ production of C(12)-HSL in AnAOB-based applications.

Aggregate Size and Architecture Determine Microbial Activity Balance for One-Stage Partial Nitritation and Anammox

Aerobic ammonium-oxidizing bacteria (AerAOB) and anoxic ammonium-oxidizing bacteria (AnAOB) cooperate in partial nitritation/anammox systems to remove ammonium from wastewater. In this process, large granular microbial aggregates enhance the performance, but little is known about granulation so far. In this study, three suspended-growth oxygen-limited autotrophic nitrification-denitrification (OLAND) reactors with different inoculation and operation (mixing and aeration) conditions, designated reactors A, B, and C, were used. The test objectives were (i) to quantify the AerAOB and AnAOB abundance and the activity balance for the different aggregate sizes and (ii) to relate aggregate morphology, size distribution, and architecture putatively to the inoculation and operation of the three reactors. A nitrite accumulation rate ratio (NARR) was defined as the net aerobic nitrite production rate divided by the anoxic nitrite consumption rate. The smallest reactor A, B, and C aggregates were nitrite sources (NARR, >1.7). Large reactor A and C aggregates were granules capable of autonomous nitrogen removal (NARR, 0.6 to 1.1) with internal AnAOB zones surrounded by an AerAOB rim. Around 50% of the autotrophic space in these granules consisted of AerAOB- and AnAOB-specific extracellular polymeric substances. Large reactor B aggregates were thin film-like nitrite sinks (NARR, <0.5) in which AnAOB were not shielded by an AerAOB layer. Voids and channels occupied 13 to 17% of the anoxic zone of AnAOB-rich aggregates (reactors B and C). The hypothesized granulation pathways include granule replication by division and budding and are driven by growth and/or decay based on species-specific physiology and by hydrodynamic shear and mixing.

A low volumetric exchange ratio allows high autotrophic nitrogen removal in a sequencing batch reactor

Sequencing batch reactors (SBRs) have several advantages, such as a lower footprint and a higher flexibility, compared to biofilm based reactors, such as rotating biological contactors. However, the critical parameters for a fast start-up of the nitrogen removal by oxygen-limited autotrophic nitrification/denitrification (OLAND) in a SBR are not available. In this study, a low critical minimum settling velocity (0.7 m h −1) and a low volumetric exchange ratio (25%) were found to be essential to ensure a fast start-up, in contrast to a high critical minimum settling velocity (2 m h −1) and a high volumetric exchange ratio (40%) which yielded no successful start-up. To prevent nitrite accumulation, two effective actions were found to restore the microbial activity balance between aerobic and anoxic ammonium-oxidizing bacteria (AerAOB and AnAOB). A daily biomass washout at a critical minimum settling velocity of 5 m h −1 removed small aggregates rich in AerAOB activity, and the inclusion of an anoxic phase enhanced the AnAOB to convert the excess nitrite. This study showed that stable physicochemical conditions were needed to obtain a competitive nitrogen removal rate of 1.1 g N L −1 d −1.

Nitrogen Removal from Digested Black Water by One-Stage Partial Nitritation and Anammox

This study assessed the technical feasibility to treat digested black water from vacuum toilets (> 1000 mg NH4(+)-N L(-1)) in a lab-scale oxygen-limited autotrophic nitrification/denitrification (OLAND) rotating biological contactor. After an adaptation period of 2.5 months, a stable nitrogen removal rate of ca. 700 mg N L(-1) d(-1) was reached over the subsequent 5 months. Suppression of the nitrite oxidizing bacteria at free ammonia levels above 3 mg N L(-1) resulted in a nitrogen removal efficiency of 76%. The favorable ratios of both organic and inorganic carbon to nitrogen guaranteed endured anammox activity and sufficient buffering capacity, respectively. Quantitative FISH showed that aerobic and anoxic ammonium-oxidizing bacteria (AerAOB and AnAOB) made up 43 and 8% of the biofilm, respectively. Since a part of the AerAOB was probably present in anoxic biofilm zones; their specific ammonium conversion was very low, in contrast to the high specific AnAOB activity. DGGE analysis showed that the dominant AerAOB and AnAOB species were resistant to the transition from synthetic medium to digested black water. This study demonstrates high-rate nitrogen removal from digested black water by one-stage partial nitritation and anammox, which will allow a significant decrease in operational costs compared to conventional nitrification/ denitrification.

Autotrophic nitrogen removal in sequencing batch biofilm reactors at different oxygen supply modes

Conventional nitrification-denitrification treatment is a common way to treat nitrogen in wastewater, but this process is costly for low COD/N wastewaters due to the addition of air and external carbon-source. However, ammonia may alternatively be converted to dinitrogen gas by autotrophic bacteria utilizing aerobically autotrophically produced nitrite as an electron acceptor under anoxic conditions. Lab-scale sequencing batch biofilm reactors (SBBRs) inoculated with normal nitrifying sludge were employed to study the potential of an oxygen-limited autotrophic nitrification-denitrification process initiated with typical nitrifying sludge for treating a synthetic ammonia wastewater devoid of organic carbon in one step. The ring-laced fibrous carrier (length 0.32 m, surface area 3.4 m2/m) was fixed vertically in a 3 L reactor. Two different air supply modes were applied:continuous aeration to control dissolved oxygen at 1.5 mg/L and intermittent aeration. High nitrogen removals of more than 50% were obtained in both SBBRs. At an ammonia loading of 0.882 gm N/m2-day [hydraulic retention time (HRT) of 24 hr], the SBBR continuously aerated to 1.5 mg DO/L had slightly higher nitrogen removal (64%) than the intermittently alternated SBBR (55%). The main form of residual nitrogen in the effluent was ammonia, at concentrations of 25 mg/L and 37 mg N/L in continuous and intermittent aeration SBBRs, respectively. Ammonia was completely consumed when ammonia loading was reduced to 0.441 gm N/m2-day [HRT extended to 48 hr]. The competitive use of nitrite by aerobic nitrite oxidizing bacteria (ANOB) with anaerobic ammonia-oxidizing bacteria (anammox bacteria) during the expanded aeration period under low remaining ammonia concentration resulted in higher nitrate production and lower nitrogen loss in the continuous aeration SBBR than in the intermittent aeration SBBR. The nitrogen removal efficiencies in SBBRs with continuous and alternating aerated were 80% and 86% respectively. Specific microorganisms in the biofilm were characterized using fluorescence in situ hybridization. Aerobic ammonia-oxidizing bacteria (AAOB) occurred side by side with putative anammox bacteria (cells hybridizing with probe AMX820) throughout the biofilm, though ANOB were rarely detected.

Innovative process scheme for removal of organic matter, phosphorus and nitrogen from pig manure

Disposal of pig manure often requires treatment with respect to environmental legislations. In this study different processes for reduction of the organic matter (anaerobic digestion, effluent separation by decanter centrifugation, membrane microfiltration, post-digestion in upflow anaerobic sludge blanket (UASB) reactor, partial oxidation), nitrogen (oxygen-limited autotrophic nitrification–denitrification, OLAND) and phosphorus (phosphorus removal by precipitation as struvite, PRS) from pig manure were tested. Results obtained showed that microfiltration was unsuitable for pig manure treatment. PRS treated effluent was negatively affecting the further processing of the pig manure in UASB, and was therefore not included in the final process flow scheme. In a final scheme (PIGMAN concept) combination of the following successive process steps was used: thermophilic anaerobic digestion with sequential separation by decanter centrifuge, post-digestion in UASB reactor, partial oxidation and finally OLAND process. This combination resulted in reduction of the total organic, nitrogen and phosphorus contents by 96%, 88%, and 81%, respectively.

Reactivation of aerobic and anaerobic ammonium oxidizers in OLAND biomass after long-term storage

The biomass of an oxygen-limited autotrophic nitrification/denitrification (OLAND) biofilm reactor was preserved in various ways to find a storage method for both aerobic and anaerobic ammonium-oxidizing bacteria (AerAOB and AnAOB). Storage occurred at -20 degrees C with and without glycerol as cryoprotectant and at 4 and 20 degrees C with and without nitrate as redox buffer. After 2 and 5 months, reactivation of AerAOB and AnAOB was achieved with the biomass stored at 4 degrees C with and without nitrate and at 20 degrees C with nitrate. Moreover, the presence of the AerAOB and AnAOB was confirmed with fluorescent in situ hybridization (FISH). Preservation in a nitrate environment resulted in a lag phase for the AnAOB reactivation. The supplied nitrate was denitrified during storage, and a real-time polymerase chain reaction with nitrifying and denitrifying genes allowed to estimate that at least 1.0 to 6.0% of the OLAND biofilm consisted of denitrifiers. It was concluded that reactivation after long-term storage is possible and that preservation at 4 degrees C without nitrate addition is the recommended storage technique. The possibility to store OLAND biomass will facilitate research on AnAOB and can overcome larger-scale start-up and inhibition problems of novel nitrogen processes involving AnAOB.

New Aspects of Microbial Nitrogen Transformations in the Context of Wastewater Treatment – A Review

AbstractOver the past few years, new technologies for nitrogen removal have been developed mainly because of the increasing financial costs of the traditional wastewater treatment technologies. Newly discovered pathways, like the anaerobic oxidation of ammonium (ANAMMOX), and uses for nitrogen removal technologies are under discussion. Processes and technologies such as: Partial nitrification; Single reactor systems for High Ammonium Removal Over Nitrite (SHARON); Anammox; Aerobic/anoxic deammonification; Oxygen Limited Autotrophic Nitrification‐Denitrification (OLAND); Completely Autotrophic Nitrogen Removal Over Nitrite (CANON); wetland based systems, all have a high potential for nitrogen removal. However, the pathways of nitrogen transformation processes are very complex. An understanding of how various environmental factors affect these processes and a sound knowledge of existing, worldwide experience pertaining to these novel technologies are the key if the nitrogen removal rates are to be improved and success is to be realized in full‐scale applications. This review describes the present knowledge of the new treatment technologies for wastewater with high nitrogen loads. Special emphasis is given to the influence of environmental factors and the reactor configuration on the nitrogen transformation process and microbial activity.

Oxygen-limited autotrophic nitrification–denitrification (OLAND) in a rotating biological contactor treating high-salinity wastewater

A lab-scale rotating biological contactor (RBC) reactor operated under OLAND conditions was slowly adapted during 178 days to increasing salt concentrations going up to 30 g NaCl L −1. The reactor performed well during this experimental period. However, the removal capacity of the reactor was lower under high-salinity conditions. A removal efficiency of 84% was achieved at a N loading rate of 725 mg N L −1 d −1 and a salt concentration of 30 g L −1. The effect of salt shock loading and adaptation to 30 g NaCl L −1 on the specific nitritation and anammox activity of the biomass was investigated in short-term batch experiments. A salt shock loading of 30 g L −1 caused a 43% decrease in specific nitritation activity and 96% loss of specific anammox activity compared to reference biomass (not exposed to salt). The salt-adapted biomass (3–4 weeks) showed a specific nitritation activity that was 23% lower, and a specific anammox activity that was 58% lower, compared to the reference biomass. Overall, these results demonstrate that the OLAND process can have the potential to treat ammonium-rich brines after adaptation to high salinity.

Denitrification pathways and rates in the sandy sediments of the Georgia continental shelf, USA.

Denitrification in continental shelf sediments has been estimated to be a significant sink of oceanic fixed nitrogen (N). The significance and mechanisms of denitrification in organic-poor sands, which comprise 70% of continental shelf sediments, are not well known. Core incubations and isotope tracer techniques were employed to determine processes and rates of denitrification in the coarse-grained, sandy sediments of the Georgia continental shelf. In these sediments, heterotrophic denitrification was the dominant process for fixed N removal. Processes such as coupled nitrification-denitrification, anammox (anaerobic ammonium oxidation), and oxygen-limited autotrophic nitrification-denitrification were not evident over the 24 and 48 h time scale of the incubation experiments. Heterotrophic denitrification processes produce 22.8–34.1 μmole N m-2 d-1 of N2 in these coarse-grained sediments. These denitrification rates are approximately two orders of magnitude lower than rates determined in fine-grained shelf sediments. These lower rates may help reconcile unbalanced marine N budgets which calculate global N losses exceeding N inputs.

Start-up of autotrophic nitrogen removal reactors via sequential biocatalyst addition.

A procedure for start-up of oxygen-limited autotrophic nitrification-denitrification (OLAND) in a lab-scale rotating biological contactor (RBC) is presented. In this one-step process, NH4+ is directly converted to N2 without the need for an organic carbon source. The approach is based on a sequential addition of two types of easily available biocatalyst to the reactor during start-up: aerobic nitrifying and anaerobic, granular methanogenic sludge. The first is added as a source of aerobic ammonia-oxidizing bacteria (AAOB), the second as a possible source of planctomycetes including anaerobic ammonia-oxidizing bacteria (AnAOB). The initial nitrifying biofilm serves as a matrix for anaerobic cell incorporation. By subsequently imposing oxygen limitation, one can create an optimal environment for autotrophic N removal. In this way, N removal of about 250 mg of N L(-1) d(-1) was achieved after 100 d treating a synthetic NH4+-rich wastewater. By gradually imposing higher loads on the reactor, the N elimination could be increased to about 1.8 g of N L(-1) d(-1) at 250 d. The resulting microbial community was compared with that of the inocula using general bacterial and AAOB- and planctomycete-specific PCR primers. Subsequently, the RBC reactor was shown to treat a sludge digestor effluent under suboptimal and strongly varying conditions. The RBC biocatalyst was also submitted to complete absence of oxygen in a fixed-film bioreactor (FFBR) and proved able to remove NH4+ with NO2- as electron acceptor (maximal 434 mg of NH4+-N (g of VSS)(-1) d(-1) on day 136). DGGE and real-time PCR analysis demonstrated that the RBC biofilm was dominated by members of the genus Nitrosomonas and close relatives of Kuenenia stuttgartiensis, a known AnAOB. The latter was enriched during FFBR operation, but AAOB were still present and the ratio planctomycetes/AAOB rRNA gene copies was about 4.3 after 136 d of reactor operation. Whether this relates to an active role of AAOB in the anoxic N removal process remains to be solved.