Robbert Kleerebezem

Nitrous oxide emission during wastewater treatment

Nitrous oxide (N(2)O), a potent greenhouse gas, can be emitted during wastewater treatment, significantly contributing to the greenhouse gas footprint. Measurements at lab-scale and full-scale wastewater treatment plants (WWTPs) have demonstrated that N(2)O can be emitted in substantial amounts during nitrogen removal in WWTPs, however, a large variation in reported emission values exists. Analysis of literature data enabled the identification of the most important operational parameters leading to N(2)O emission in WWTPs: (i) low dissolved oxygen concentration in the nitrification and denitrification stages, (ii) increased nitrite concentrations in both nitrification and denitrification stages, and (iii) low COD/N ratio in the denitrification stage. From the literature it remains unclear whether nitrifying or denitrifying microorganisms are the main source of N(2)O emissions. Operational strategies to prevent N(2)O emission from WWTPs are discussed and areas in which further research is urgently required are identified.

Enrichment of a Mixed Bacterial Culture with a High Polyhydroxyalkanoate Storage Capacity

Polyhydroxyalkanoates (PHAs) are microbial storage polymers that attract interest as bioplastics. PHAs can be produced with open mixed cultures if a suitable enrichment step based on the ecological role of PHA is used. An acetate-fed sequencing batch reactor operated with 1 day biomass residence time and with feast-famine cycles of 12 h was used to enrich a mixed culture of PHA producers. In subsequent fed-batch experiments under growth limiting conditions, the enriched mixed culture produced PHA up to a cellular content of 89 wt % within 7.6 h (average rate of 1.2 g/g/h). The PHA produced from acetate was the homopolymer polyhydroxybutyrate. The culture was dominated by a Gammaproteobacterium that showed little similarity on 16S rRNA level with known bacteria (<90% sequence similarity). The mixed culture process for PHA production does not require aseptic conditions. Waste streams rather than pure substrates could be used as raw materials.

Nitrogen Removal by a Nitritation-Anammox Bioreactor at Low Temperature

ABSTRACT Currently, nitritation-anammox (anaerobic ammonium oxidation) bioreactors are designed to treat wastewaters with high ammonium concentrations at mesophilic temperatures (25 to 40°C). The implementation of this technology at ambient temperatures for nitrogen removal from municipal wastewater following carbon removal may lead to more-sustainable technology with energy and cost savings. However, the application of nitritation-anammox bioreactors at low temperatures (characteristic of municipal wastewaters except in tropical and subtropical regions) has not yet been explored. To this end, a laboratory-scale (5-liter) nitritation-anammox sequencing batch reactor was adapted to 12°C in 10 days and operated for more than 300 days to investigate the feasibility of nitrogen removal from synthetic pretreated municipal wastewater by the combination of aerobic ammonium-oxidizing bacteria (AOB) and anammox. The activities of both anammox and AOB were high enough to remove more than 90% of the supplied nitrogen. Multiple aspects, including the presence and activity of anammox, AOB, and aerobic nitrite oxidizers (NOB) and nitrous oxide (N2O) emission, were monitored to evaluate the stability of the bioreactor at 12°C. There was no nitrite accumulation throughout the operational period, indicating that anammox bacteria were active at 12°C and that AOB and anammox bacteria outcompeted NOB. Moreover, our results showed that sludge from wastewater treatment plants designed for treating high-ammonium-load wastewaters can be used as seeding sludge for wastewater treatment plants aimed at treating municipal wastewater that has a low temperature and low ammonium concentrations.

Nitrification expanded: discovery, physiology and genomics of a nitrite-oxidizing bacterium from the phylum Chloroflexi

Nitrite-oxidizing bacteria (NOB) catalyze the second step of nitrification, a major process of the biogeochemical nitrogen cycle, but the recognized diversity of this guild is surprisingly low and only two bacterial phyla contain known NOB. Here, we report on the discovery of a chemolithoautotrophic nitrite oxidizer that belongs to the widespread phylum Chloroflexi not previously known to contain any nitrifying organism. This organism, named Nitrolancetus hollandicus, was isolated from a nitrifying reactor. Its tolerance to a broad temperature range (25–63 °C) and low affinity for nitrite (Ks=1 mM), a complex layered cell envelope that stains Gram positive, and uncommon membrane lipids composed of 1,2-diols distinguish N. hollandicus from all other known nitrite oxidizers. N. hollandicus grows on nitrite and CO2, and is able to use formate as a source of energy and carbon. Genome sequencing and analysis of N. hollandicus revealed the presence of all genes required for CO2 fixation by the Calvin cycle and a nitrite oxidoreductase (NXR) similar to the NXR forms of the proteobacterial nitrite oxidizers, Nitrobacter and Nitrococcus. Comparative genomic analysis of the nxr loci unexpectedly indicated functionally important lateral gene transfer events between Nitrolancetus and other NOB carrying a cytoplasmic NXR, suggesting that horizontal transfer of the NXR module was a major driver for the spread of the capability to gain energy from nitrite oxidation during bacterial evolution. The surprising discovery of N. hollandicus significantly extends the known diversity of nitrifying organisms and likely will have implications for future research on nitrification in natural and engineered ecosystems.

Anammox growth on pretreated municipal wastewater

Autotrophic nitrogen removal from municipal wastewater enables development of energy autarkic wastewater treatment plants. In this study we report the evaluation of the anammox process in a granular sludge fluidized bed lab-scale reactor continuously fed with the actual effluent of the A-stage of the WWTP of Dokhaven, Rotterdam. The reactor was anoxic, and nitrite was dosed continuously to support anammox activity only. The system was operated for more than ten months at temperatures between 20 and 10 °C. COD was also consumed during the process, but heterotrophs could not outcompete anammox bacteria. Volumetric N-removal rates obtained were comparable or higher than those of conventional N-removal systems, with values higher than 0.4 g-N L(-1) d(-1) when operated at 10 °C. The biomass specific N-removal rate at 10 °C was on average 50±7 mg-N g-VSS(-1) d(-1) during the last month of operations, almost two times higher than previously reported activities at this temperature. FISH analysis revealed that the dominant anammox species was Candidatus Brocadia Fulgida throughout the experimentation. Evidence for growth of anammox bacteria at mainstream conditions was demonstrated for the entire temperature range tested (10-20 °C), and new granules were shown to be actively formed and efficiently retained in the system.

Emission of nitrous oxide and nitric oxide from a full-scale single-stage nitritation-anammox reactor

At a full-scale single-stage nitritation-anammox reactor, off-gas measurement for nitric oxide (NO) and nitrous oxide (N(2)O) was performed. NO and N(2)O are environmental hazards, imposing the risk of improving water quality at the cost of deteriorating air quality. The emission of NO during normal operation of a single-stage nitritation-anammox process was 0.005% of the nitrogen load while the N(2)O emission was 1.2% of the nitrogen load to the reactor, which is in the same range as reported emission from other full-scale wastewater treatment plants. The emission of both compounds was strongly coupled. The concentration of NO and N(2)O in the off-gas of the single-stage nitritation-anammox reactor was rather dynamic and clearly responded to operational variations. This exemplifies the need for time-dependent measurement of NO and N(2)O emission from bioreactors for reliable emission estimates. Nitrite accumulation clearly resulted in increased NO and N(2)O concentrations in the off-gas, yielding higher emission levels. Oxygen limitation resulted in a decrease in NO and N(2)O emission, which was unexpected as oxygen limitation is generally assumed to cause increased emissions in nitrogen converting systems. Higher aeration flow dramatically increased the NO emission load and also seemed to increase the N(2)O emission, which stresses the importance of efficient aeration control to limit NO and N(2)O emissions.

Influence of the C/N ratio on the performance of polyhydroxybutyrate (PHB) producing sequencing batch reactors at short SRTs

Many waste streams that are suitable substrates for mixed culture bioplastic (polyhydroxyalkanoate, PHA) production are nutrient limited and may need to be supplemented to allow sufficient growth of PHA accumulating bacteria. The scope of this study was to investigate the necessity of nutrient supplementation for the enrichment of an efficient PHA producing mixed culture. We studied the influence of different degrees of carbon and nitrogen limitation on the performance of an acetate-fed feast-famine sequencing batch reactor (SBR) employed to enrich PHA storing bacteria. The microbial reaction rates in the SBR showed a shift with a change in the limiting substrate: high acetate uptake rates were found in carbon-limited SBRs (medium C/N ratios 6-13.2 Cmol/Nmol), while nitrogen-limited SBRs (medium C/N ratios 15-24 Cmol/Nmol) were characterized by high ammonia uptake rates. Biomass in strongly nitrogen-limited SBRs had higher baseline PHA contents in the SBR, but carbon-limited SBRs resulted usually in biomass with higher maximal PHA storage capacities. The PHA storage capacity in a nitrogen-limited SBR operated at 0.5 d SRT decreased significantly over less than 5 months operation. For the microbial selection and biomass production stage of a PHA production process carbon limitation seems thus favourable and nutrient deficient wastewaters may consequently require supplementation with nutrients for the selection of a stable PHA storing biomass with a high storage capacity.

Glycerol fermentation by (open) mixed cultures: a chemostat study.

Glycerol is an important byproduct of bioethanol and biodiesel production processes. This study aims to evaluate its potential application in mixed culture fermentation processes to produce bulk chemicals. Two chemostat reactors were operated in parallel, one fed with glycerol and the other with glucose. Both reactors operated at a pH of 8 and a dilution rate of 0.1 h−1. Glycerol was mainly converted into ethanol and formate. When operated under substrate limiting conditions, 60% of the substrate carbon was converted into ethanol and formate in a 1:1 ratio. This product spectrum showed sensitivity to the substrate concentration, which partly shifted towards 1,3‐propanediol and acetate in a 2:1 ratio at increasing substrate concentrations. Glucose fermentation mainly generated acetate, ethanol and butyrate. At higher substrate concentrations, acetate and ethanol were the dominant products. Co‐fermentations of glucose–glycerol were performed with both mixed cultures, previously cultivated on glucose and on glycerol. The product spectrum of the two experiments was very similar: the main products were ethanol and butyrate (38% and 34% of the COD converted, respectively). The product spectrum obtained for glucose and glycerol fermentation could be explained based on the general metabolic pathways found for fermentative microorganisms and on the metabolic constraints: maximization of the ATP production rate and balancing the reducing equivalents involved. Biotechnol. Biotechnol. Bioeng. 2008;100: 1088–1098.

Segregation of Biomass in Cyclic Anaerobic/Aerobic Granular Sludge Allows the Enrichment of Anaerobic Ammonium Oxidizing Bacteria at Low Temperatures

A cyclic anaerobic/aerobic bubble column reactor was run for 420 days to study the competition for nitrite between nitrite oxidizing bacteria (NOB) and anaerobic ammonium oxidizing bacteria (Anammox) at low temperatures. An anaerobic feeding period with nitrite and ammonium in the influent followed by an aerated period was applied resulting in a biomass specific conversion rate of 0.18 ± 0.02 [gN(2) - N · gVSS(-1)· day(-1)] when the dissolved oxygen concentration was maintained at 1.0 mgO(2) · L(-1). An increase in white granules was observed in the reactor which were mainly located at the top of the settled sludge bed, whereas red granules were located at the bottom. FISH, activity tests, and qPCR techniques revealed that red biomass was dominated by Anammox bacteria and white granules by NOB. Granules from the top of the sludge bed were smaller and therefore had a higher aerobic volume fraction, a lower density, and consequently a slower settling rate. Sludge was manually removed from the top of the settled sludge bed to selectively remove NOB which resulted in an increased overall biomass specific N-conversion rate of 0.32 ± 0.02 [gN(2) - N · gVSS(-1) · day(-1)]. Biomass segregation in granular sludge reactors gives an extra opportunity to select for specific microbial groups by applying a different SRT for different microbial groups.

Pilot-scale evaluation of anammox-based mainstream nitrogen removal from municipal wastewater

Autotrophic nitrogen removal in the mainstream wastewater treatment process is suggested to be a prerequisite of energy autarkic wastewater treatment plants (WWTP). Whilst the application of anammox-related technologies in the side-stream is at present state of the art, the feasibility of this energy-efficient process at mainstream conditions is still under development. Lower operating temperature and ammonium concentration, together with required high nitrogen removal efficiency, represent the main challenges to face in order to reach this appealing new frontier of the wastewater treatment field. In this study, we report the evaluation of the process in a plug-flow granular sludge-based pilot-scale reactor (4 m3) continuously fed with the actual effluent of the A-stage of the WWTP of Dokhaven, Rotterdam. The one-stage partial nitritation–anammox system was operated for more than 10 months at 19 ± 1°C. Observed average N-removal and ammonium conversion rates were comparable or higher than those of conventional N-removal systems, with 182 ± 46 and 315 ± 33 mg-N L−1 d−1, respectively. Biochemical oxygen demand was also oxidized in the system with an average removal efficiency of 90%. Heterotrophic biomass grew preferentially in flocs and was efficiently washed out of the system. Throughout the experimentation, the main bottleneck was the nitritation process that resulted in nitrite-limiting conditions for the anammox conversion. Anammox bacteria were able to grow under mainstream WWTP conditions and new granules were formed and efficiently retained in the system.